Yossi Dagon

Nutritional Status, Cognition, and Survival A NEW ROLE FOR LEPTIN AND AMP KINASE

Although adequate nutrition is essential for optimal neural activity and survival, mild energy restriction may improve cognition and prolong longevity. Energy status is monitored by the cellular AMP-activated protein kinase (AMPK) system, whereas leptin regulates total energy balance. We investigated the roles of AMPK and leptin in cognition and survival under diet restriction (DR). Hippocampal AMPK activity increases with energy restriction. Modest activation (DR to 60%) induces neurogenesis and improves cognition. However, DR to 40% augmented AMPK activity, reduced cognition and catecholamines, and increased neural apoptosis and mortality. Leptin signaling is preserved only in DR to 60%, countering the effects of AMPK “overactivation” by preventing neuroapoptosis, restoring noradrenergic activity and behavioral performance, and increasing longevity. The balance between leptin and AMPK is crucial in determining neuronal fate, cognitive ability, and survival. Should these findings extend to Man, then controlled activation of AMPK may improve neurodegenerative diseases, and leptin may have a new role in treating stress-associated malnutrition.

Endocannabinoids affect neurological and cognitive function in thioacetamide-induced hepatic encephalopathy in mice

Endocannabinoids function as neurotransmitters and neuromodulators in the central nervous system via specific receptors and apparently have a neuroprotective role. We assumed that the endocannabinoid system could be involved in the pathogenesis of hepatic encephalopathy (HE), a neuropsychiatric syndrome due to liver disease. We used a mouse model of a thioacetamide induced fulminant hepatic failure. We found that the levels of the endocannabinoid 2-arachidonoyl-glycerol (2-AG) were elevated in the brain. Treatment with either 2-AG or with the CB1 receptor antagonist, SR141716A, improved a neurological score, activity and cognitive function. Activation of the CB2 receptor by a selective agonist, HU308, also improved the neurological score. 2-AG activity could be blocked with the specific CB2 receptor antagonist SR144528A. The CB1 receptor agonist noladin ether was inactive. We conclude that the endocannabinoid system may play an important role in the pathogenesis of HE. Modulation of this system either by exogenous agonists specific for the CB2 receptors or possibly also by antagonists to the CB1 receptors may have therapeutic potential.

Cannabinoids ameliorate cerebral dysfunction following liver failure via AMP-activated protein kinase

Hepatic encephalopathy (HE) is a neuropsychiatry disorder of complex pathogenesis caused by acute or chronic liver failure. We studied the etiology of cerebral dysfunction in a murine model of HE induced by either bile duct ligation or thioacetamide administration. We report that stimulation of cerebral AMP‐activated protein kinase (AMPK), a major intracel‐lular energy sensor, is a compensatory response to liver failure. This function of AMPK is regulated by endo‐cannabinoids. The cannabinoid system controls systemic energy balance via the cannabinoid receptors CB‐1 and CB‐2. Under normal circumstances, AMPK activity is mediated by CB‐1 while CB‐2 is barely detected. However, CB‐2 is strongly stimulated in response to liver failure. Administration of ∆9‐tetrahy‐drocannabinol (THC) augmented AMPK activity and restored brain function in WT mice but not in their CB‐2 KO littermates. These results suggest that HE is a disease of energy flux. CB‐2 signaling is a cerebral stress response mechanism and makes AMPK a promising target for its treatment by modulating the canna‐binoid system.—Dagon, Y., Avraham, Y., Ilan, Y., Mechoulam, R., Berry, E. M. Cannabinoids ameliorate cerebral dysfunction following liver failure via AMP‐activated protein kinase. FASEB J. 21, 2431–2441 (2007)

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells.

Background: Diminished activity of the enzyme AMP-activated protein kinase (AMPK) is associated with impaired insulin signaling. Results: Protein Kinase (PK)C/D1 activation inhibits AMPKα2 via Ser491 phosphorylation; PKD1 inhibition prevents this in skeletal muscle cells. Conclusion: PKD1 is a novel upstream AMPK-kinase that phosphorylates AMPK on Ser491 and regulates insulin signaling. Significance: PKD1 inhibition may be a novel strategy for improving insulin sensitivity. AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser491 in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser491 that plays a negative role in insulin signaling in muscle cells.

Essential Roles of Raf/Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway, YY1, and Ca2+ Influx in Growth Arrest of Human Vascular Smooth Muscle Cells by Bilirubin

Background: Bilirubin circulates throughout the human cardiovascular system. Its interaction with the vascular wall is not well known. Results: Bilirubin alters Raf/ERK/MAPK pathway, cellular transcription factor YY1 location, and calcium-dependent YY1 proteolysis in human vascular cells. Conclusion: At high physiological levels bilirubin, inhibits cell growth, inhibits proliferation, and does not cause apoptosis. Significance: The observations provide opportunities for prevention and treatment of cardiovascular diseases. The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.

Complement factors are secreted in human follicular fluid by granulosa cells and are possible oocyte maturation factors

Aims:u2002 In this study, we identify components of the complement system present in human follicular fluid that affect oocyte development and maturation.