Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKβ, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes.     

LKB1 is the upstream kinase in the AMP-activated protein kinase cascade

Inactivating mutations in the protein kinase LKB1 lead to a dominantly inherited cancer in humans termed Peutz-Jeghers syndrome. The role of LKB1 is unclear, and only one target for LKB1 has been identified in vivo [3]. AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a pivotal role in energy homeostasis. AMPK may have a role in protecting the body from metabolic diseases including type 2 diabetes, obesity, and cardiac hypertrophy. We previously reported the identification of three protein kinases (Elm1, Pak1, and Tos3 [9]) that lie upstream of Snf1, the yeast homologue of AMPK. LKB1 shares sequence similarity with Elm1, Pak1, and Tos3, and we demonstrated that LKB1 phosphorylates AMPK on the activation loop threonine (Thr172) within the catalytic subunit and activates AMPK in vitro [9]. Here, we have investigated whether LKB1 corresponds to the major AMPKK activity present in cell extracts. AMPKK purified from rat liver corresponds to LKB1, and blocking LKB1 activity in cells abolishes AMPK activation in response to different stimuli. These results identify a link between two protein kinases, previously thought to lie in unrelated, distinct pathways, that are associated with human diseases.     

Resistin is a key mediator of glucose-dependent insulinotropic polypeptide (GIP) stimulation of lipoprotein lipase (LPL) activity in adipocytes

Studies on the physiological roles of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) have largely focused on its insulinotropic action and ability to regulate beta-cell mass. In previous studies on the stimulatory effect of GIP on adipocyte lipoprotein lipase (LPL), a pathway was identified involving increased phosphorylation of protein kinase B (PKB) and reduced phosphorylation of LKB1 and AMP-activated protein kinase (AMPK). The slow time of onset of the responses suggested that GIP may have induced release of an intermediary molecule, and the current studies focused on the possible contribution of the adipokine resistin. In differentiated 3T3-L1 adipocytes, GIP, in the presence of insulin, increased resistin secretion through a pathway involving p38 mitogen-activated protein kinase (p38 MAPK) and the stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK). The other major incretin hormone, glucagon-like peptide-1 (GLP-1), exhibited no significant effects. Chronic elevation of circulating GIP levels in the Vancouver Diabetic Fatty (VDF) Zucker rat resulted in increases in circulating resistin levels and activation of p38 MAPK or SAPK/JNK in epididymal fat tissue, suggesting the existence of identical pathways in vivo as well as in vitro. Administration of resistin to 3T3-L1 adipocytes mimicked the effects of GIP on the PKB/LKB1/AMPK/LPL pathway: increasing phosphorylation of PKB, reducing levels of phosphorylated LKB1 and AMPK, and increasing LPL activity. Knockdown of resistin using RNA interference attenuated the effect of GIP on the PKB/LKB1/AMPK/LPL pathway in 3T3-L1 adipocytes, supporting a role for resistin as a mediator.     

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.     

Regulation of glucose-dependent insulinotropic polypeptide release by protein in the rat

Glucose-dependent insulinotropic polypeptide (GIP) release has been demonstrated predominantly after ingestion of carbohydrate and fat. These studies were conducted to determine the effects of protein on GIP expression in the rat. Whereas no significant changes in duodenal mucosal GIP mRNA levels were detected in response to peptone, the duodenal GIP concentration increased from 8.4+/-1.5 to 19.8+/-3.2 ng GIP/mg protein at 120 min (P<0.01). Plasma GIP levels also increased from 95+/-5.2 pg/ml to a peak of 289+/-56.1 pg/ml at 120 min (P<0.01). To determine whether the effects of protein on GIP were due to stimulation of acid secretion, rats were pretreated with 10 mg/kg omeprazole, after which mucosal and plasma GIP concentrations were partially attenuated. To further examine the effects of luminal acid, rats were administered intraduodenal 0.1 M HCl for 120 min, which significantly enhanced GIP expression. These studies indicate that nutrient protein provides a potent stimulus for GIP expression in the rat, an effect that occurs at the posttranslational level and may be mediated in part through the acid-stimulatory properties of protein. The effects of acid on GIP are consistent with a role for GIP as an enterogastrone in the rat.     

A novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide: protein kinase A-dependent endocytosis

The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (K(ATP)) and calcium-activated (K(CA)) potassium channels have been extensively studied, but less is known about the role of voltage-gated (K(V)) potassium channels in pancreatic beta-cells. The incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion by potentiating events underlying membrane depolarization and exerting direct effects on exocytosis. In the present study, we identified a novel role for GIP in regulating K(V)1.4 channel endocytosis. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of K(V)1.4 via activation of protein kinase A (PKA). Using mutant forms of K(V)1.4 with Ala-Ser/Thr substitutions in a potential PKA phosphorylation site, C-terminal phosphorylation was shown to be linked to GIP-mediated current amplitude decreases. Proteinase K digestion and immunocytochemical studies on mutant K(V)1.4 localization following GIP stimulation demonstrated phosphorylation-dependent rapid endocytosis of K(V)1.4. Expression of K(V)1.4 protein was also demonstrated in human beta-cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells. Transient overexpression in INS-1 beta-cells (clone 832/13) of wild-type (WT) K(V)1.4, or a T601A mutant form resistant to PKA phosphorylation, resulted in reduced glucose-stimulated insulin secretion; WT K(V)1.4 overexpression potentiated GIP-induced insulin secretion, whereas this response was absent in T601A cells. These results strongly support an important novel role for GIP in regulating K(V)1.4 cell surface expression and modulation of A-type potassium currents, which is likely to be critically important for its insulinotropic action.     

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.     

Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity

AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPKalpha(1)/alpha(2) on Ser(485/491) in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPKalpha at its primary activation site, Thr(172) (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335-5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPKalpha at Thr(172), which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491). To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPKalpha, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPKalpha at Thr(172), despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491) and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPKalpha at Thr(172). Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491).     

Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein

Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. The human GIP gene with the human A-fibrinopeptide sequence was synthesized and linked to the Staphylococcus aureus protein A gene in the vector pRIT2T. This plasmid was expressed in Escherichia coli, and the resulting fusion protein consisted of three domains: protein A for ease of purification, fibrinopeptide sequence for thrombin cleavage and human GIP. The GIP was subsequently cleaved from the fusion protein with -thrombin. The identity of the recombinant human GIP was confirmed by SDS-PAGE, ELISA, HPLC and amino-terminal amino acid sequence analysis. This recombinant product was shown to have comparable insulinotropic activity to porcine GIP in the isolated perfused pancreas.     

Inhibition of insulin, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) secretion by octreotide has no effect on post-heparin plasma lipoprotein lipase activity.

This study examines the immediate effect of modulating postprandial insulin and insulinotropic hormone (glucose-dependent insulinotropic polypeptide, GIP; glucagon-like peptide-1, GLP-1) secretion on the activation of lipoprotein lipase (LPL) in six lean and six obese age-matched women. Subjects were given, on two separate occasions, 340 kcal of carbohydrate alone or combined with an IV infusion of octreotide, (100 microg infusion from 30 min before the meal for 150 min). Post-heparin LPL activity (10,000 U) was measured on each occasion 120 minutes post-carbohydrate. Following oral carbohydrate postprandial plasma insulin levels were significantly higher in obese subjects than in lean (p < 0.01). Glucose tolerance was slightly impaired in obese subjects. Insulin, GIP and GLP-1 secretion post-carbohydrate was markedly reduced by octreotide in lean and obese subjects. LPL activity was similar in the two groups after carbohydrate administration and was unaffected by octreotide. Inhibition of postprandial insulin, GIP and GLP-1 secretion acutely did not reduce post-heparin LPL activity either in lean or obese subjects.     

SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation

SIRT1, a histone/protein deacetylase, and AMP-activated protein kinase (AMPK) are key enzymes responsible for longevity and energy homeostasis. We examined whether a mechanistic connection exists between these molecules that involves the major AMPK kinase LKB1. Initial studies demonstrated that LKB1 is acetylated in cultured (HEK293T) cells, mouse white adipose tissue, and rat liver. In the 293T cells, SIRT1 overexpression diminished lysine acetylation of LKB1 and concurrently increased its activity, cytoplasmic/nuclear ratio, and association with the LKB1 activator STRAD. In contrast, short hairpin RNA for SIRT1, where studied, had opposite effects on these parameters. Mass spectrometric analysis established that acetylation of LKB1 occurs on multiple, but specific, lysine residues; however, only mutation of lysine 48 to arginine, which mimics deacetylation, reproduced all of the effects of activated SIRT1. SIRT1 also affected downstream targets of LKB1. Thus its overexpression increased AMPK and acetyl-CoA carboxylase phosphorylation, and conversely, RNA interference-mediated SIRT1 knockdown reduced AMPK phosphorylation and that of another LKB1 target MARK1. Consistent with the results in cultured cells, total LKB1 lysine acetylation was decreased by 60% in the liver of 48-h starved rats compared with starved-refed rats, and this was associated with modest but significant increases in both LKB1 and AMPK activities. These results suggest that LKB1 deacetylation is regulated by SIRT1 and that this in turn influences its intracellular localization, association with STRAD, kinase activity, and ability to activate AMPK.     

Activation of protein phosphatase 2A by palmitate inhibits AMP-activated protein kinase

Elevated levels of free fatty acids contribute to cardiovascular diseases, but the mechanisms remain poorly understood. The present study was aimed to determine if free fatty acid inhibits the AMP-activated kinase (AMPK). Exposure of cultured bovine aortic endothelial cells (BAECs) to palmitate (0.4 mM) but not to palmitoleic or oleic acid (0.4 mM) for 40 h significantly reduced the Thr(172) phosphorylation of AMPK-alpha without altering its protein expression or the phosphorylation of LKB1-Ser(428), a major AMPK kinase in BAECs. Further, in LKB1-deficient cells, palmitate suppressed AMPK-Thr(172) implying that the inhibitory effects of palmitate on AMPK might be independent of LKB1. In contrast, 2-bromopalmitate, a non-metabolizable analog of palmitate, did not alter the phosphorylation of AMPK and acetyl-CoA carboxylase. Further, palmitate significantly increased the activity of protein phosphatase (PP)2A. Inhibition of PP2A with either okadaic acid, a selective PP2A inhibitor, or PP2A small interference RNA abolished palmitate-induced inhibition on AMPK-Thr(172) phosphorylation. Exposure of BAECs to C(2)-ceramide, a cell-permeable analog of ceramide, mimicked the effects of palmitate. Conversely, fumonisin B1, which selectively inhibits ceramide synthase and decreases de novo formation of ceramide, abolished the effects of palmitate on both PP2A and AMPK. Inhibition of AMPK in parallel with increased PP2A activity was founded in C57BL/6J mice fed with high fat diet (HFD) rich in palmitate but not in mice fed with HFD rich in oleate. Moreover, inhibition of PP2A with PP2A-specific siRNA but not scrambled siRNA reversed HFD-induced inhibition on the phosphorylation of AMPK-Thr(172) and endothelial nitric-oxide synthase (eNOS)-Ser(1177) in mice fed with high fat diets. Taken together, we conclude that palmitate inhibits the phosphorylation of both AMPK and endothelial nitric-oxide synthase in endothelial cells via ceramide-dependent PP2A activation.     

Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.     

Endurance training increases LKB1 and MO25 protein but not AMP-activated protein kinase kinase activity in skeletal muscle

LKB1 complexed with MO25 and STRAD has been identified as an AMP-activated protein kinase kinase (AMPKK). We measured relative LKB1 protein abundance and AMPKK activity in liver (LV), heart (HT), soleus (SO), red quadriceps (RQ), and white quadriceps (WQ) from sedentary and endurance-trained rats. We examined trained RQ for altered levels of MO25 protein and LKB1, STRAD, and MO25 mRNA. LKB1 protein levels normalized to HT (1 +/- 0.03) were LV (0.50 +/- 0.03), SO (0.28 +/- 0.02), RQ (0.32 +/- 0.01), and WQ (0.12 +/- 0.03). AMPKK activities in nanomoles per gram per minute were HT (79 +/- 6), LV (220 +/- 9), SO (22 +/- 2), RQ (29 +/- 2), and WQ (42 +/- 4). Training increased LKB1 protein in SO, RQ, and WQ (P < 0.05). LKB1 protein levels after training (%controls) were SO (158 +/- 17), RQ (316 +/- 17), WQ (191 +/- 27), HT (106 +/- 2), and LV (104 +/- 7). MO25 protein after training (%controls) was 595 +/- 71. Training did not affect AMPKK activity. MO25 but not LKB1 or STRAD mRNA increased with training (P < 0.05). Trained values (%controls) were MO25 (164 +/- 22), LKB1 (120 +/- 16), and STRAD (112 +/- 17). LKB1 protein content strongly correlated (r = 0.93) with citrate synthase activity in skeletal muscle (P < 0.05). In conclusion, endurance training markedly increased skeletal muscle LKB1 and MO25 protein without increasing AMPKK activity. LKB1 may be playing multiple roles in skeletal muscle adaptation to endurance training.     

Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism

Hormone-sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP-activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin-dependent protein kinase II also phosphorylates hormone-sensitive lipase predominantly at this site, whilst cyclic-GMP-dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone-sensitive lipase by the cyclic-AMP-dependent and cyclic-GMP-dependent protein kinases, indicating that site-2 phosphorylation may have an antilipolytic role in vivo.     

Nicotine-induced activation of AMP-activated protein kinase inhibits fatty acid synthase in 3T3L1 adipocytes: a role for oxidant stress

Recent studies suggest that the AMP-activated protein kinase (AMPK) acts as a major energy sensor and regulator in adipose tissues. The objective of this study was to investigate the role of AMPK in nicotine-induced lipogenesis and lipolysis in 3T3L1 adipocytes. Exposure of 3T3L1 adipocytes to smoking-related concentrations of nicotine increased lipolysis and inhibited fatty acid synthase (FAS) activity in a time- and dose-dependent manner. The effects of nicotine on FAS activity were accompanied by phosphorylation of both AMPK (Thr(172)) and acetyl-CoA carboxylase (ACC; Ser(79)). Nicotine-induced AMPK phosphorylation appeared to be mediated by reactive oxygen species based on the finding that nicotine significantly increased superoxide anions and 3-nitrotyrosine-positive proteins, exogenous peroxynitrite (ONOO(-)) mimicked the effects of nicotine on AMPK, and N-acetylcysteine (NAC) abolished nicotine-enhanced AMPK phosphorylation. Inhibition of AMPK using either pharmacologic (insulin, compound C) or genetic means (overexpression of dominant negative AMPK; AMPK-DN) abolished FAS inhibition induced by nicotine or ONOO(-). Conversely, activation of AMPK by pharmacologic (nicotine, ONOO(-), metformin, and AICAR) or genetic (overexpression of constitutively active AMPK) means inhibited FAS activity. Notably, AMPK activation increased threonine phosphorylation of FAS, and this effect was blocked by adenovirus encoding dominant negative AMPK. Finally, AMPK-dependent FAS phosphorylation was confirmed by (32)P incorporation into FAS in adipocytes. Taken together, our results strongly suggest that nicotine, via ONOO(-) activates AMPK, resulting in enhanced threonine phosphorylation and consequent inhibition of FAS.     

Effects of 3-phosphoglycerate and other metabolites on the activation of AMP-activated protein kinase by LKB1-STRAD-MO25

Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase (AMPKK). The LKB1-STE-related adaptor (STRAD)-mouse protein 25 (MO25) complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study, we tested an array of metabolites including, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate (3-PG), glucose 1-phosphate, glucose 1,6-bisphosphate, ADP, carnitine, acetylcarnitine, IMP, inosine, and ammonia for allosteric regulation. ADP inhibited both AMPK and LKB1-STRAD-MO25 actions, but probably is not important physiologically because of the low free ADP inside the muscle fiber. We found that 3-PG stimulated LKB1-STRAD-MO25 activity and allowed for increased AMPK phosphorylation. 3-PG did not stimulate LKB1-STRAD-MO25 activity toward the peptide substrate LKB1tide. These results have identified 3-PG as an AMPK-specific regulator of AMPK phosphorylation and activation by LKB1-STRAD-MO25.     

LKB1, a protein kinase regulating cell proliferation and polarity

LKB1 is a serine-threonine protein kinase mutated in patients with an autosomal dominantly inherited cancer syndrome predisposing to multiple benign and malignant tumours, termed Peutz-Jeghers syndrome. Since its discovery in 1998, much research has focused on identification and characterisation of its cellular roles and analysing how LKB1 might be regulated. In this review we discuss exciting recent advances indicating that LKB1 functions as a tumour suppressor perhaps by controlling cell polarity. We also outline the current understanding of the molecular mechanisms by which LKB1 is regulated in vivo, through interaction with other proteins as well as by protein phosphorylation and prenylation.