Oxygen radicals derived from xanthine oxidase (XO) are important mediators of the cellular injury associated with reperfusion of ischemic intestine, stomach, liver, kidney, and pancreas. XO exists in nonischemic tissue predominantly as xanthine dehydrogenase (XDH) and converts to oxygen radical-producing XO with ischemia. Grinding intestine under liquid nitrogen and placing the powder in phosphate buffer (pH 7.0) containing thiol reductants and protease inhibitors adequately preserved total XDH + XO activity and the percentage in the oxidase form (%XO) for 24 h. Total activity in nonischemic intestine ranged from 374 nmol.min-1.g-1 in duodenum to 138 nmol.min-1.g-1 in ileum, while XO activity was approximately 19% of total activity throughout the entire small intestine. The rate of XDH conversion to XO during normothermic ischemia varied only slightly throughout the intestine, increasing 13% per hour to 34, 46, and 61% XO after 1, 2, and 3 h of ischemia, respectively. Our results contrast with previous reports where XDH conversion to XO occurred within 60 s ischemia but are consistent with physiological and morphological evidence of ischemic injury and provide further support for involvement of XO in intestinal injury associated with ischemia.

In this study, we utilized genetic and cell biological approaches to evaluate potential functions for the AMPKα C-terminus. We identify a critical new function for the carboxy-terminal amino acids of AMPKα in vivo, which affects AMPKα subcellular localization, phosphorylation, and ultimately organismal viability.

Mammalian brain connectivity requires the coordinated production and migration of billions of neurons and the formation of axons and dendrites. The LKB1/Par4 kinase is required for axon formation during cortical development in vivo partially through its ability to activate SAD-A/B kinases. LKB1 is a master kinase phosphorylating and activating at least 11 other serine/threonine kinases including the metabolic sensor AMP-activated protein kinase (AMPK), which defines this branch of the kinome. A recent study using a gene-trap allele of the β1 regulatory subunit of AMPK suggested that AMPK catalytic activity is required for proper brain development including neurogenesis and neuronal survival. We used a genetic lossof-function approach producing AMPKα1/α2-null cortical neurons to demonstrate that AMPK catalytic activity is not required for cortical neurogenesis, neuronal migration, polarization, or survival. However, we found that application of metformin or AICAR, potent AMPK activators, inhibit axogenesis and axon growth in an AMPK-dependent manner. We show that inhibition of axon growth mediated by AMPK overactivation requires TSC1/2-mediated inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. Our results demonstrate that AMPK catalytic activity is not required for early neural development in vivo but its overactivation during metabolic stress impairs neuronal polarization in a mTOR-dependent manner.A MP-activated kinase (AMPK) is a heterotrimeric serine/ threonine protein kinase composed of one catalytic subunit (encoded by α1 or α2 genes in mammals) and two regulatory subunits β and γ (encoded by β1 or β2 genes and γ1, γ2, or γ3 genes, respectively) (1-3). AMPK is an important metabolic sensor, activated by various forms of metabolic stress including low ATP: AMP ratios. AMPK has been implicated in a range of cell biological functions including cell polarity, autophagy, apoptosis, and cell migration (2-9). A recent study (10) suggested that the regulatory subunit, AMPKβ1, is critical for normal neurogenesis, neuronal differentiation, and neuronal survival during cortical development. However, to date there is no published evidence reporting the consequence of a genetic loss of function for the catalytic activity of mammalian AMPK in the mammalian nervous system. To assess the role of AMPKα during cortical development, we used transgenic mice that were ubiquitously inactivated for the AMPKα1 gene (AMPKα1 −/− ) (11) and conditionally inactivated for AMPKα2 (AMPKα2 F/F ) (12, 13). AMPKα2 was selectively deleted using the Emx1 Cre mouse line, which induces recombination only in dorsal telencephalic progenitors giving rise to all pyramidal projection neurons in the cortex, but not in ventral telencephalon-derived cortical GABAergic interneurons, which constitutes ∼25% of all cortical neurons (14). Surprisingly, we found no obvious defect of neurogenesis, neuronal migration, axon formation, or neuronal survival in AMPKα1/2-null cortex compared with control mice. On the basis of the profound di...

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Class II histone deacetylases (HDACs) act as repressors of cardiac hypertrophy, an adaptative response of the heart characterized by a reprogramming of fetal cardiac genes. Prolonged hypertrophy often leads to dilated cardiomyopathy and heart failure. Upstream endogenous regulators of class II HDACs that regulate hypertrophic growth are just beginning to emerge. Herewedemonstratethatthe␦Bisoformofcalcium/calmodulindependent protein kinase II (CaMKII␦B), known to promote cardiac hypertrophy, transmits signals specifically to HDAC4 but not other class II HDACs. CaMKII␦B efficiently phosphorylates both a glutathione S-transferase (GST)-HDAC4 fragment spanning amino acids 207-311 and full-length FLAG-HDAC4 but not the equivalents in HDAC5. Although previous studies in skeletal muscle cells have shown that HDAC4 lacking serine 246 cannot be phosphorylated by CaMKI/IV, a similar mutant is still phosphorylated by CaMKII␦B. Importantly, mutation of serine 210 to alanine totally abolishes phosphorylation of the GST fragment and significantly reduces phosphorylation of full-length HDAC by CaMKII␦B. RNA interference knockdown of CaMKII␦B prevents the effects of hypertrophic stimuli. Overexpression of CaMKII␦B in primary neonatal cardiomyocytes increases the activity of the Mef2 transcription factor and completely rescues HDAC4-mediated repression of MEF2 but only partially rescues inhibition by HDAC5 or the HDAC4 S210A mutant. CaMKII␦B strongly interacts with HDAC4 in cells but not with HDAC5. These results demonstrate that CaMKII␦B preferentially targets HDAC4, and this involves serine 210. These findings identify HDAC4 as a specific downstream substrate of CaMKII␦B in cardiac cells and have broad applications for the signaling pathways leading to cardiac hypertrophy and heart failure.