Curcumin is an early-acting stage-specific inducer of extended functional longevity in Drosophila.

Larval feeding with curcumin induces an extended health span with significantly increased median and maximum longevities in the adult fly. This phenotype is diet insensitive and shows no additive effect on longevity when combined with an adult dietary restriction (DR) diet, suggesting that curcumin and DR operate via the same or overlapping pathways for this trait. This treatment significantly slows the aging rate so that it is comparable with that of genetically selected long lived animals. The larval treatment also enhances the adult animal's geotactic activity in an additive manner with DR, suggesting that curcumin and DR may use different pathways for different traits. Feeding the drug to adults during only the health span also results in a significantly extended health span with increased median and maximum life span. This extended longevity phenotype is induced only during these stage-specific periods. Feeding adults with the drug over their whole life results in a weakly negative effect on median longevity with no increase in maximum life span. There are no negative effects on reproduction, although larval curcumin feeding increases development time, and also apparently accelerates the normal late-life neuromuscular degeneration seen in the legs. Gene expression data from curcumin-fed larvae shows that the TOR pathway is inhibited in the larvae and the young to midlife adults, although several other genes involved in longevity extension are also affected. These data support the hypothesis that curcumin acts as if it is a DR mimetic nutraceutical. These data also suggest that the search for DR mimetics may be enhanced by the use of stage-specific screening of candidate molecules.

Perilipin targets a novel pool of lipid droplets for lipolytic attack by hormone-sensitive lipase.

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.

Secretion without membrane fusion: porocytosis.

We have recently proposed a mechanism to describe secretion, a fundamental process in all cells. That hypothesis, called porocytosis, embodies all available data and encompasses both forms of secretion, i.e., vesicular and constitutive. The current accepted view of exocytotic secretion involves the physical fusion of vesicle and plasma membranes; however, that hypothesized mechanism does not fit all available physiological data. Energetics of apposed lipid bilayers do not favor unfacilitated fusion. We consider that calcium ions (e.g., 10(-4) to 10(-3) M calcium in microdomains when elevated for 1 ms or less), whose mobility is restricted in space and time, establish salt bridges among adjacent lipid molecules. This establishes transient pores that span both the vesicle and plasma membrane lipid bilayers; the diameter of this transient pore would be approximately 1 nm (the diameter of a single lipid molecule). The lifetime of the transient pore is completely dependent on the duration of sufficient calcium ion levels. This places the porocytosis hypothesis for secretion squarely in the realm of the physical and physical chemical interactions of calcium and phospholipids and places mass action as the driving force for release of secretory material. The porocytosis hypothesis that we propose satisfies all of the observations and provides a framework to integrate our combined knowledge of vesicular and constitutive secretion.

Porocytosis: a transient pore array secretes the neurotransmitter packet.

The porocytosis hypothesis is based on the arrayed nature of synaptic vesicles which forms the anatomical functional unit of secretion. The presynaptic array and the postsynaptic array of receptors form a synaptomere which is the unit of transmission. A transient increase in calcium ions, triggered by an action potential, activates all pores of the array to pulse transmitter. The array insures transmission while permitting a frequency dependent amount of secretion. Therefore the amount of secretion is variable which permits plasticity. Secretion from the array has the property of immediate synaptic plasticity whereas a change in array size would change synaptic strength. The robust nature of the array insures fidelity of transmission, a frequency dependent dynamic signature of transmission giving the property of immediate plasticity; and, a change in array size yields a change in synaptic strength for long term reliability.

The synaptic bouton acts like a salt shaker.

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.

Mastoparan-induced insulin secretion from insulin-secreting betaTC3 and INS-1 cells: evidence for its regulation by Rho subfamily of G proteins.

Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet beta-cells, presumably via activation of trimeric G proteins. Herein, we used Clostridial toxins, which selectively modify and inactivate the Rho subfamily of G proteins, to examine whether mastoparan-induced insulin secretion also involves activation of these signaling proteins. Mastoparan, but not mastoparan 17 (an inactive analog of mastoparan), significantly stimulated insulin secretion from betaTC3 and INS-1 cells. Preincubation of betaTC3 cells with either Clostridium difficille toxin B, which inactivates Rho, Cdc42, and Rac, or Clostridium sordellii toxin, which inactivates Ras, Rap, and Rac, markedly attenuated the mastoparan-induced insulin secretion, implicating Rac in this phenomenon. Mastoparan-stimulated insulin secretion was resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of Rho G proteins (e.g. Rac), suggesting that mastoparan induces direct activation of Rac via GTP/GDP exchange. This was confirmed by a pull-down assay that quantifies the binding of activated (i.e. GTP-bound) Rac to p21-activated kinase. However, glucose-induced insulin secretion from these cells was abolished by toxin B or GGTI-2147, suggesting that the geranylgeranylation step is critical for glucose-stimulated secretion. Mastoparan significantly increased the translocation of cytosolic Rac and Cdc42 to the membrane fraction. Confocal light microscopy revealed a substantial degree of colocalization of Rac (and, to a lesser degree, Cdc42) with insulin in beta-cells exposed to mastoparan. Further, stable expression of a dominant negative (N17Rac) form of Rac into INS-1 cells resulted in a significant reduction in mastoparan-stimulated insulin secretion from these cells. Taken together, our findings implicate Rho G proteins, specifically Rac, in mastoparan-induced insulin release.

Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates.

The small GTPase Rab2 requires atypical protein kinase C iota/lambda (PKCiota/lambda) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCiota/lambda in the early secretory pathway. A peptide corresponding to the unique PKCiota/lambda pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCiota/lambda, beta-COP, and p53/p58. Because Rab2, beta-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCiota/lambda (W274K). Both the pseudosubstrate peptide and kinase-dead PKCiota/lambda in tandem with Rab2 caused sustained membrane association of PKCiota/lambda, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCiota/lambda was reversed by PKCiota/lambda wild type. These combined results indicate that PKCiota/lambda is essential for protein transport in the early secretory pathway and suggest that PKCiota/lambda kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.

Porocytosis: secretion from small and medium-diameter vesicles and vesicle arrays without membrane fusion.

We have recently proposed a mechanism to describe secretion, a fundament process in all cells. That hypothesis, called porocytosis, embodies all available data, and encompasses both forms of secretion, i.e., vesicular and constitutive. The current accepted view of exocytotic secretion involves the physical fusion of vesicle- and plasma membranes. However, that hypothesized mechanism does not fit all available physiological data (Silver et al., 2001; Kriebel et al., 2001). Energetics of apposed lipid bilayers do not favor unfacilitated fusion. Calcium ion levels are elevated in microdomains at levels of 10(-4)-10(-3)M for 1 ms or less, with the calcium ions showing limited lateral mobility at the site of secretion (Llinas et al., 1992, Silver et al., 1994). We consider that calcium ions, whose mobility is restricted in space and time, establish "salt-bridges" among adjacent lipid molecules, and establishes transient pores that span the vesicle and plasma membrane lipid bilayers; the lifetime of that transient pore being completely dependent on duration of sufficient calcium ion levels.