Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that chronic increase of cytosolic calcium concentration in hepatocytes upon obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome-lysosome fusion in liver, preventing accumulation of protein inclusions and lipid droplets and suppressing inflammation and insulin resistance. As calcium channel blockers have been safely used in clinics for the treatment of hypertension for more than thirty years, our results suggest they may be a safe therapeutic option for restoring autophagic flux and treating metabolic pathologies in obese patients.

Membrane fusion is a process that intimately involves both proteins and lipids. Although the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipidbinding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid-binding-deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics, suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid-generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A-lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events. INTRODUCTIONMembrane fusion is a process that underlies compartmentalization within all eukaryotic cells, and allows for the many critical and diverse physiological functions in higher organisms. Despite the essential and ubiquitous nature of this process, a considerable energetic expenditure is required to overcome the electrostatic repulsion between opposing lipid bilayers and to deform and ultimately rupture these bilayers (Chernomordik and Kozlov, 2003;Cohen and Melikyan, 2004). As a result, substantial effort has been placed on defining the molecular machinery that overcomes this energetic barrier to accomplish regulated and rapid membrane fusion. SNARE (soluble n-ethylmaleimide-sensitive fusion factor attachment protein receptor) proteins have now been identified as the minimal protein machinery required for membrane fusion (Jahn and Scheller, 2006). Their critical role is supported by multiple lines of evidence, including that SNARE proteins are sufficient to drive membrane fusion when reconstituted into liposomes in vitro (Weber et al., 1998) and that cleavage of SNARE proteins by clostridial toxins (Schiavo et al., 1992; Blasi et al., 1993a,b), as well as genetic mutations resulting in loss of SNARE protein function (Broadie et al., 1995;Littleton et al., 1998;Saifee et al., 1998), strongly inhibit neurotransmitter release. Currently, the role of SNARE proteins in membrane fusion is believed to be predominantly mechanical. During neurotransmitter release, nucleation and zippering of a highly stable SNARE core complex formed f...

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca 2؉ . One of these priming steps involves the maintenance of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P 2 ) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P 2 is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P 2 important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P 2 that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4,5-P 2 and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P 2 , showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca 2؉ entry and suggest that plasma membrane PtdIns-4,5-P 2 is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P 2 in some manner alters calcium channel function in chromaffin cells.

Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p protein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is derived from SNAP receptor (the soluble Nethylmaleimide-sensitive fusion protein)). However, this interaction inhibits vesicle SNARE interactions with syntaxin that are required for secretory vesicles to achieve competency for membrane fusion. As such, regulation of the interaction between Munc18a and syntaxin 1a may provide an important mechanism controlling secretory responsiveness. Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts directly with Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation. We have now demonstrated that Cdk5 is capable of phosphorylating Munc18a in vitro within a preformed Munc18a⅐syntaxin 1a heterodimer complex and that this results in the disassembly of the complex. Using sitedirected mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr 574 . Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase activity. Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine secretion from chromaffin cells, an effect not observed with the inactive analogue iso-olomoucine. The effects of olomoucine were independent of calcium influx as evidenced by secretory inhibition in permeabilized chromaffin cells and in cells under whole-cell voltage clamp. Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotinic agonist-induced secretory responses. Our data suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin 1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a and secretory responsiveness.