Structural basis of AMPK regulation by small molecule activators.

AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

5'-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes.

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4 min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and ß-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-ß-D-ribofuranoside), agents that mimic 'metabolic stress'. Inactivation of AMPK by adrenaline was abolished by 1 µM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK ß-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (ß-Ser24, ß-Ser108 or ß-Ser182) suggesting that another site(s) is targeted.

Dietary fatty acids make a rapid and substantial contribution to VLDL-triacylglycerol in the fed state.

Exaggerated postprandial lipemia is associated with coronary heart disease and type II diabetes, yet few studies have examined the effect of sequential meals on lipoprotein metabolism. We have used 13C-labeled fatty acids to trace the incorporation of fatty acid derived from a meal into apolipoprotein B-100 (apoB-100)-containing lipoproteins and plasma nonesterified fatty acids (NEFA) following two consecutive meals. Healthy volunteers (n=8) were given breakfast labeled with [1-(13)C]palmitic acid, eicosapentaenoic acid, and docosahexaenoic acid, followed 5 h later by lunch containing [1-(13)C]oleic acid. Blood samples were taken over a 9-h period. ApoB-100-containing lipoproteins were isolated by immunoaffinity chromatography. Chylomicron-triacylglycerol (TG) concentrations peaked at 195 min following breakfast but at 75 min following lunch (P<0.001). VLDL-TG concentrations, in contrast, rose to a broad peak after breakfast and then fell steadily after lunch. Breakfast markers followed chylomicron-TG concentrations and appeared in plasma NEFA with a similar profile, whereas [1-(13)C]oleic acid peaked 2 h after lunch in plasma TG and NEFA. Breakfast markers appeared steadily in VLDL, peaking 1-3 h after lunch, whereas [1-(13)C]oleic acid was still accumulating in VLDL at 9 h. Around 17% of VLDL-TG originated from recent dietary fat 5 h after breakfast, and around 40% at the end of the experiment. We conclude that there is rapid flux of fatty acids from the diet into endogenous pools. Further study of these processes may open up new targets for intervention to reduce VLDL-TG concentrations and postprandial lipemia.

Selective partitioning of dietary fatty acids into the VLDL TG pool in the early postprandial period.

Circulating triacylglycerol (TG) arises mainly from dietary fat. However, little is known about the entry of dietary fat into the major TG pool, very low-density lipoprotein (VLDL) TG. We used a novel method to study the specific incorporation of dietary fatty acids into postprandial VLDL TG in humans. Eight healthy volunteers (age 25.4 +/- 2.2 years, body mass index 22.1 +/- 2.3 kg/m2) were fed a mixed meal containing 30 g fish oil and 600 mg [1-13C]palmitic acid. Chylomicrons and VLDL were separated using immunoaffinity against apolipoprotein B-100. The fatty acid composition of lipoproteins was analyzed by gas chromatography/mass spectrometry. [1-13C]palmitic acid started to appear in VLDL TG 3 h after meal intake, and a similar delay was observed for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Approximately 20% of dietary fatty acids entered the VLDL TG pool 6 h after meal intake. DHA was clearly overincorporated into this pool compared with [1-13C]palmitic acid and EPA. This seemed to depend on a marked elevation of this fatty acid in the nonesterified fatty acid pool. In summary, the contribution of dietary fatty acids to early postprandial VLDL TG is substantial. The role of DHA in VLDL TG production will require further investigation.