Acute regulation of 5'-AMP-activated protein kinase by long-chain fatty acid, glucose and insulin in rat primary adipocytes.

Palmitate increased AMPK (5'-AMP-activated protein kinase) activity, glucose utilization and 2-DOG (2-deoxyglucose) transport in rat adipocytes. All three effects were blocked by the AMPK inhibitor Compound C, leading to the conclusion that in response to an increase in long-chain NEFA (non-esterified fatty acid) concentration AMPK mediated an enhancement of adipocyte glucose transport, thereby providing increased glycerol 3-phosphate for FA (fatty acid) esterification to TAG (triacylglycerol). Activation of AMPK in response to palmitate was not due to an increase in the adipocyte AMP:ATP ratio. Glucose decreased AMPK activity and effects of palmitate and glucose on AMPK activity were antagonistic. While insulin had no effect on basal AMPK activity insulin did decrease AMPK activity in the presence of palmitate and also decreased the percentage effectiveness of palmitate to increase the transport of 2-DOG. It is suggested that activation of adipocyte AMPK by NEFA, as well as decreasing the activity of hormone-sensitive lipase, could modulate adipose tissue dynamics by increasing FA esterification and, under certain circumstances, FA synthesis.

Inactivation of the AMP-activated protein kinase by glucose in cardiac myocytes: a role for the pentose phosphate pathway.

Incubation of adult rat cardiac myocytes with increasing glucose concentrations decreased phosphorylation (αThr172) and activity of AMPK (AMP-activated protein kinase). The effect could be demonstrated without measurable changes in adenine nucleotide contents. The glucose effect was additive to the decrease in AMPK activity caused by insulin, was attenuated by adrenaline, was not mimicked by glucose analogues, lactate or pyruvate and was not due to changes in myocyte glycogen content. AMPK activity was decreased by xylitol and PMS (phenazine methosulfate) and was increased by the glucose-6-phosphate dehydrogenase inhibitor DHEA (dehydroepiandrosterone) and by thiamine. PMS and DHEA respectively, increased and decreased CO2 formation by the PPP (pentose phosphate pathway). AMPK activity was inversely related to the myocyte content of Xu5P (xylulose 5-phosphate), an intermediate of the non-oxidative arm of the PPP. Endothall, an inhibitor of PP2A (protein phosphatase 2A), abolished the glucose effect on AMPK activity. Further studies are needed to define the 'active component' that mediates the glucose effect and whether its site of action is PP2A.

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.

Malonyl-CoA, a key signaling molecule in mammalian cells.

Malonyl-CoA can be formed within the mitochondria, peroxisomes, and cytosol of mammalian cells. Besides being an intermediate in the pathways of de novo fatty acid biosynthesis and fatty acid elongation, malonyl-CoA has an important signaling function through its allosteric inhibition of carnitine palmitoyltransferase 1, the enzyme that normally exerts flux control over mitochondrial beta-oxidation. Malonyl-CoA is rapidly turned over in mammalian cells, and the activities of acetyl-CoA carboxylase and malonyl-CoA decarboxylase are important determinants of its cytosolic concentration. It is now recognized that malonyl-CoA participates in a diverse range of physiological or pathological responses and systems. These include the ketogenic response of the liver to fasting and diabetes, carbohydrate versus fat fuel selection in muscle tissues, metabolic changes in muscle during contracture, alterations in fatty acid metabolism during cardiac ischemia and postischemic reperfusion, stimulation of B cell insulin secretion by glucose, and the hypothalamic control of appetite.

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.

A new mouse mutant, skijumper.

Low blood sugar levels are a well-known cause of severe illness and often death in newborn humans, especially those that are small for age. Few of the causes of neonatal hypoglycemia are known, and many remain to be found. We describe a novel mouse mutant, skijumper (skimp), in which pups, despite feeding well, have low levels of glucose and develop opisthotonos, followed by death typically within a few days after birth. Genetic mapping studies have localized the lesion to a approximately 1 cM interval on mouse Chromosome (Chr) 7 between D7Mit318 and D7Mit93. We have carried out extensive analysis to define the phenotype and its likely cause. In addition to low blood glucose, affected skijumper mice have lowglycogen and ketone levels. Mass spectrometric analysis of blood samples has excluded major defects in amino acid metabolism. Initial biochemical analyses suggested a defect in ketogenesis as one possible cause of this phenotype. However, measurements of levels and activities of carnitine, carnitine palmitoyl transferases, and other enzymes involved in ketogenesis, along with studies of mitochondrial structure and function, did not demonstrate significant differences between skijumper, unaffected littermates, and control wild-type mice. These results indicate that abnormal enzyme activity in known pathways does not appear to be the primary biochemical lesion in skijumper. The skijumper may be a new valuable model for studying and understanding one type of neonatal morbidity and death.