Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia.

Basal cells are by definition located on the basolateral side of several epithelia, and they have never been observed reaching the lumen. Using high-resolution 3D confocal imaging, we report that basal cells extend long and slender cytoplasmic projections that not only reach toward the lumen but can cross the tight junction barrier in some epithelia of the male reproductive and respiratory tracts. In this way, the basal cell plasma membrane is exposed to the luminal environment. In the epididymis, in which luminal acidification is crucial for sperm maturation and storage, these projections contain the angiotensin II type 2 receptor (AGTR2). Activation of AGTR2 by luminal angiotensin II, increases proton secretion by adjacent clear cells, which are devoid of AGTR2. We propose a paradigm in which basal cells scan and sense the luminal environment of pseudostratified epithelia and modulate epithelial function by a mechanism involving crosstalk with other epithelial cells.

The mitochondrial complex V-associated large-conductance inner membrane current is regulated by cyclosporine and dexpramipexole.

Inefficiency of oxidative phosphorylation can result from futile leak conductance through the inner mitochondrial membrane. Stress or injury may exacerbate this leak conductance, putting cells, and particularly neurons, at risk of dysfunction and even death when energy demand exceeds cellular energy production. Using a novel method, we have recently described an ion conductance consistent with mitochondrial permeability transition pore (mPTP) within the c-subunit of the ATP synthase. Excitotoxicity, reactive oxygen species-producing stimuli, or elevated mitochondrial matrix calcium opens the channel, which is inhibited by cyclosporine A and ATP/ADP. Here we show that ATP and the neuroprotective drug dexpramipexole (DEX) inhibited an ion conductance consistent with this c-subunit channel (mPTP) in brain-derived submitochondrial vesicles (SMVs) enriched for F1FO ATP synthase (complex V). Treatment of SMVs with urea denatured extramembrane components of complex V, eliminated DEX- but not ATP-mediated current inhibition, and reduced binding of [(14)C]DEX. Direct effects of DEX on the synthesis and hydrolysis of ATP by complex V suggest that interaction of the compound with its target results in functional conformational changes in the enzyme complex. [(14)C]DEX bound specifically to purified recombinant b and oligomycin sensitivity-conferring protein subunits of the mitochondrial F1FO ATP synthase. Previous data indicate that DEX increased the efficiency of energy production in cells, including neurons. Taken together, these studies suggest that modulation of a complex V-associated inner mitochondrial membrane current is metabolically important and may represent an avenue for the development of new therapeutics for neurodegenerative disorders.

Quantitative exploration of the contribution of settlement, growth, dispersal and grazing to the accumulation of natural marine biofilms on antifouling and fouling-release coatings.

The accumulation of microbial biofilms on ships' hulls negatively affects ship performance and efficiency while also playing a role in the establishment of even more detrimental hard-fouling communities. However, there is little quantitative information on how the accumulation rate of microbial biofilms is impacted by the balance of the rates of cell settlement, in situ production (ie growth), dispersal to surrounding waters and mortality induced by grazers. These rates were quantified on test panels coated with copper-based antifouling (AF) or polymer-based fouling-release (FR) coatings by using phospholipids as molecular proxies for microbial biomass. The results confirmed the accepted modes of efficacy of these two types of coatings. In a more extensive set of experiments with only the FR coatings, it was found that seasonally averaged cellular production rates were 1.5 ± 0.5 times greater than settlement and the dispersal rates were 2.7 ± 0.8 greater than grazing. The results of this study quantitatively describe the dynamic balance of processes leading to the accumulation of microbial biofilm on coatings designed for ships' hulls.

Mitochondrial leak metabolism induces the Spemann-Mangold Organizer via Hif-1α in Xenopus.

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F1Fo ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when ß-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling.

The level of menadione redox-cycling in pancreatic ß-cells is proportional to the glucose concentration: role of NADH and consequences for insulin secretion.

Pancreatic ß-cells release insulin in response to elevation of glucose from basal (4-7mM) to stimulatory (8-16mM) levels. Metabolism of glucose by the ß-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H(2)O(2)), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H(2)O(2) inhibit insulin secretion. Menadione, which produces H(2)O(2) via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on ß-cell metabolism and insulin secretion in INS-1 832/13, a rat ß-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H(2)O(2) production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1-10µM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H(2)O(2) formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H(2)O(2) and menadione on insulin secretion.

Windows to cell function and dysfunction: signatures written in the boundary layers.

The medium surrounding cells either in culture or in tissues contains a chemical mix varying with cell state. As solutes move in and out of the cytoplasmic compartment they set up characteristic signatures in the cellular boundary layers. These layers are complex physical and chemical environments the profiles of which reflect cell physiology and provide conduits for intercellular messaging. Here we review some of the most relevant characteristics of the extracellular/intercellular space. Our initial focus is primarily on cultured cells but we extend our consideration to the far more complex environment of tissues, and discuss how chemical signatures in the boundary layer can or may affect cell function. Critical to the entire essay are the methods used, or being developed, to monitor chemical profiles in the boundary layers. We review recent developments in ultramicro electrochemical sensors and tailored optical reporters suitable for the task in hand.

Catechol metabolites of endogenous estrogens induce redox cycling and generate reactive oxygen species in breast epithelial cells.

Estrogens are major risk factors for the development of breast cancer; they can be metabolized to catechols, which are further oxidized to DNA-reactive quinones and semiquinones (SQs). These metabolites are mutagenic and may contribute to the carcinogenic activity of estrogens. Redox cycling of the SQs and subsequent generation of reactive oxygen species (ROS) is also an important mechanism leading to DNA damage. The SQs of exogenous estrogens have been shown to redox cycle, however, redox cycling and the generation of ROS by endogenous estrogens has never been characterized. In the present studies, we determined whether the catechol metabolites of endogenous estrogens, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone and 2-hydroxyestriol, can redox cycle in breast epithelial cells. These catechol estrogens, but not estradiol, estrone, estriol or 2-methoxyestradiol, were found to redox cycle and generate hydrogen peroxide (H(2)O(2)) and hydroxyl radicals in lysates of three different breast epithelial cell lines: MCF-7, MDA-MB-231 and MCF-10A. The generation of ROS required reduced nicotinamide adenine dinucleotide phosphate as a reducing equivalent and was inhibited by diphenyleneiodonium, a flavoenzyme inhibitor, indicating that redox cycling is mediated by flavin-containing oxidoreductases. Using extracellular microsensors, catechol estrogen metabolites stimulated the release of H(2)O(2) by adherent cells, indicating that redox cycling occurs in viable intact cells. Taken together, these data demonstrate that catechol metabolites of endogenous estrogens undergo redox cycling in breast epithelial cells, resulting in ROS production. Depending on the localized concentrations of catechol estrogens and enzymes that mediate redox cycling, this may be an important mechanism contributing to the development of breast cancer.

Plasma membrane electron transport in pancreatic ß-cells is mediated in part by NQO1.

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal ß-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of ß-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in ß-cells.

Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons.

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.

Review and Hypothesis: A Potential Common Link Between Glial Cells, Calcium Changes, Modulation of Synaptic Transmission, Spreading Depression, Migraine, and Epilepsy-H.

There is significant evidence to support the notion that glial cells can modulate the strength of synaptic connections between nerve cells, and it has further been suggested that alterations in intracellular calcium are likely to play a key role in this process. However, the molecular mechanism(s) by which glial cells modulate neuronal signaling remains contentiously debated. Recent experiments have suggested that alterations in extracellular H+ efflux initiated by extracellular ATP may play a key role in the modulation of synaptic strength by radial glial cells in the retina and astrocytes throughout the brain. ATP-elicited alterations in H+ flux from radial glial cells were first detected from Müller cells enzymatically dissociated from the retina of tiger salamander using self-referencing H+-selective microelectrodes. The ATP-elicited alteration in H+ efflux was further found to be highly evolutionarily conserved, extending to Müller cells isolated from species as diverse as lamprey, skate, rat, mouse, monkey and human. More recently, self-referencing H+-selective electrodes have been used to detect ATP-elicited alterations in H+ efflux around individual mammalian astrocytes from the cortex and hippocampus. Tied to increases in intracellular calcium, these ATP-induced extracellular acidifications are well-positioned to be key mediators of synaptic modulation. In this article, we examine the evidence supporting H+ as a key modulator of neurotransmission, review data showing that extracellular ATP elicits an increase in H+ efflux from glial cells, and describe the potential signal transduction pathways involved in glial cell-mediated H+ efflux. We then examine the potential role that extracellular H+ released by glia might play in regulating synaptic transmission within the vertebrate retina, and then expand the focus to discuss potential roles in spreading depression, migraine, epilepsy, and alterations in brain rhythms, and suggest that alterations in extracellular H+ may be a unifying feature linking these disparate phenomena.

Release and elementary mechanisms of nitric oxide in hair cells.

The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency-current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca(2+) current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca(2+) channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca(2+)-activated K(+) currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells.

Physiological and pharmacological characterizations of the larval Anopheles albimanus rectum support a change in protein distribution and/or function in varying salinities.

Ion regulation is a biological process crucial to the survival of mosquito larvae and a major organ responsible for this regulation is the rectum. The recta of anopheline larvae are distinct from other subfamilies of mosquitoes in several ways, yet have not yet been characterized extensively. Here we characterize the two major cell types of the anopheline rectum, DAR and non-DAR cells, using histological, physiological, and pharmacological analyses. Proton flux was measured at the basal membrane of 2%- and 50%-artificial sea water-reared An. albimanus larvae using self-referencing ion-selective microelectrodes, and the two cell types were found to differ in basal membrane proton flux. Additionally, differences in the response of that flux to pharmacological inhibitors in larvae reared in 2% versus 50% ASW indicate changes in protein function between the two rearing conditions. Finally, histological analyses suggest that the non-DAR cells are structurally suited for mediating ion transport. These data support a model of rectal ion regulation in which the non-DAR cells have a resorptive function in freshwater-reared larvae and a secretive function in saline water-reared larvae. In this way, anopheline larvae may adapt to varying salinities.

Ion trapping with fast-response ion-selective microelectrodes enhances detection of extracellular ion channel gradients.

Previously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 microm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10-55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane.

Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity.

In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O(2) consumption, cytosolic Ca(2+) concentration ([Ca(2+)](i)), and mitochondrial membrane potential (mDeltapsi) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca(2+)](i) and mDeltapsi were monitored with Fluo-4 and TMRE(+), respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca(2+)](i) followed seconds afterwards by an increase in O(2) consumption which reached a maximum level within 1-5 min. A modest increase in mDeltapsi occurred during this time period, and then, shortly before maximal O(2) consumption was reached, the mDeltapsi, as indicated by TMRE(+) fluorescence, dissipated. Maximal O(2) consumption lasted up to 5 min and then declined together with mDeltapsi and ATP levels, while [Ca(2+)](i) further increased. mDeltapsi and [Ca(2+)](i) returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca(2+)](i) increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O(2) consumption, [Ca(2+)](i), and mDeltapsi. The data obtained using this new method are consistent with a model where Ca(2+) influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.

Parkinson's disease protein DJ-1 regulates ATP synthase protein components to increase neuronal process outgrowth.

Familial Parkinson's disease (PD) protein DJ-1 mutations are linked to early onset PD. We have found that DJ-1 binds directly to the F1FO ATP synthase ß subunit. DJ-1's interaction with the ß subunit decreased mitochondrial uncoupling and enhanced ATP production efficiency while in contrast mutations in DJ-1 or DJ-1 knockout increased mitochondrial uncoupling, and depolarized neuronal mitochondria. In mesencephalic DJ-1 KO cultures, there was a progressive loss of neuronal process extension. This was ameliorated by a pharmacological reagent, dexpramipexole, that binds to ATP synthase, closing a mitochondrial inner membrane leak and enhancing ATP synthase efficiency. ATP synthase c-subunit can form an uncoupling channel; we measured, therefore, ATP synthase F1 (ß subunit) and c-subunit protein levels. We found that ATP synthase ß subunit protein level in the DJ-1 KO neurons was approximately half that found in their wild-type counterparts, comprising a severe defect in ATP synthase stoichiometry and unmasking c-subunit. We suggest that DJ-1 enhances dopaminergic cell metabolism and growth by its regulation of ATP synthase protein components.

Modulation of extracellular proton fluxes from retinal horizontal cells of the catfish by depolarization and glutamate.

Self-referencing H(+)-selective microelectrodes were used to measure extracellular proton fluxes from cone-driven horizontal cells isolated from the retina of the catfish (Ictalurus punctatus). The neurotransmitter glutamate induced an alkalinization of the area adjacent to the external face of the cell membrane. The effect of glutamate occurred regardless of whether the external solution was buffered with 1 mM HEPES, 3 mM phosphate, or 24 mM bicarbonate. The AMPA/kainate receptor agonist kainate and the NMDA receptor agonist N-methyl-D-aspartate both mimicked the effect of glutamate. The effect of kainate on proton flux was inhibited by the AMPA/kainate receptor blocker CNQX, and the effect of NMDA was abolished by the NMDA receptor antagonist DAP-5. Metabotropic glutamate receptor agonists produced no alteration in proton fluxes from horizontal cells. Depolarization of cells either by increasing extracellular potassium or directly by voltage clamp also produced an alkalinization adjacent to the cell membrane. The effects of depolarization on proton flux were blocked by 10 microM nifedipine, an inhibitor of L-type calcium channels. The plasmalemma Ca(2+/)H(+) ATPase (PMCA) blocker 5(6)-carboxyeosin also significantly reduced proton flux modulation by glutamate. Our results are consistent with the hypothesis that glutamate-induced extracellular alkalinizations arise from activation of the PMCA pump following increased intracellular calcium entry into cells. This process might help to relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons from photoreceptor synaptic terminals. Our findings argue strongly against the hypothesis that protons released by horizontal cells act as the inhibitory feedback neurotransmitter that creates the surround portion of the receptive fields of retinal neurons.

Imaging the electric field associated with mouse and human skin wounds.

We have developed a noninvasive instrument called the bioelectric field imager (BFI) for mapping the electric field between the epidermis and the stratum corneum near wounds in both mouse and human skin. Rather than touching the skin, the BFI vibrates a small metal probe with a displacement of 180 mum in air above the skin to detect the surface potential of the epidermis through capacitative coupling. Here we describe our first application of the BFI measuring the electric field between the stratum corneum and epidermis at the margin of skin wounds in mice. We measured an electric field of 177+/-14 (61) mV/mm immediately upon wounding and the field lines pointed away from the wound in all directions around it. Because the wound current flows immediately upon wounding, this is the first signal indicating skin damage. This electric field is generated at the outer surface of the epidermis by the outward flow of the current of injury. An equal and opposite current must flow within the multilayered epidermis to generate an intraepidermal field with the negative pole at the wound site. Because the current flowing within the multilayered epidermis is spread over a larger area, the current density and subsequent E field generated in that region is expected to be smaller than that measured by the BFI beneath the stratum corneum. The field beneath the stratum corneum typically remained in the 150-200 mV/mm range for 3 days and then began to decline over the next few days, falling to zero once wound healing was complete. The mean wound field strength decreased by 64+/-7% following the application of the sodium channel blocker, amiloride, to the skin near the wound and increased by 82+/-21% following the application of the Cl- channel activator, prostaglandin E2.

Characterization of optimized Na+ and Cl- liquid membranes for use with extracellular, self-referencing microelectrodes.

Self-referencing with ion-selective microelectrodes (ISMs) is a useful approach for monitoring near-real-time ion flux near single cells and across epithelia. While ISMs for H+, Ca2+, and K+ have been optimized for use with self-referencing, ISMs for two other primary inorganic ions, Na+ and Cl-, have not. In this study, we have characterized ISMs based on three Na+ ionophores (I, VI, and X) and one Cl- ionophore to assess their suitability for use with self-referencing. ISMs constructed with Na+ ionophore VI have short response times (approximately 100 ms) but possess nearly an order of magnitude less selectivity for Na+ over K+ than ISMs constructed with Na+ ionophore X. The Na+ ionophore X mixture was enhanced to give it a shorter response time while not compromising its selectivity. A Cl(-)-selective microelectrode was constructed and characterized with superior anionic selectivity compared with previously reported Cl- ISMs used with self-referencing. This Cl(-)-selective microelectrode, however, has a relatively slow response time (approximately 3 s), thus requiring changes to the self-referencing protocol. Self-referencing with these ISMs will enable near-real-time ion flux measurements for Na+ and Cl-.

Ca2+, NAD(P)H and membrane potential changes in pancreatic beta-cells by methyl succinate: comparison with glucose.

The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1). Saturation of the Ca2+ signal occurred between 5 and 6 mM MeS, while secretion reached its maximum at 15 mM, suggesting operation of a K(ATP)-channel-independent pathway. Additional responses to MeS and glucose included elevated NAD(P)H autofluorescence in INS-1 cells and islets and increases in assayed NADH and NADPH and the ATP/ADP ratio. Increased NADPH and ATP/ADP ratios occurred more rapidly with MeS, although similar levels were reached after 5 min of exposure to each fuel, whereas NADH increased more with MeS than with glucose. Reversal of MeS-induced cell depolarization by Methylene Blue completely inhibited MeS-stimulated secretion, whereas basal secretion and KCl-induced changes in these parameters were not affected. MeS had no effect on secretion or signals in the mouse islets, in contrast with glucose, possibly due to a lack of malic enzyme. The data are consistent with the common intermediates being pyruvate, cytosolic NADPH or both, and suggest that cytosolic NADPH production could account for the more rapid onset of MeS-induced secretion compared with glucose stimulation.

Physiological increases in uncoupling protein 3 augment fatty acid oxidation and decrease reactive oxygen species production without uncoupling respiration in muscle cells.

Decreased uncoupling protein (UCP)3 is associated with insulin resistance in muscle of pre-diabetic and diabetic individuals, but the function of UCP3 remains unclear. Our goal was to elucidate mechanisms underlying the negative correlation between UCP3 and insulin resistance in muscle. We determined effects of physiologic UCP3 overexpression on glucose and fatty acid oxidation and on mitochondrial uncoupling and reactive oxygen species (ROS) production in L6 muscle cells. An adenoviral construct caused a 2.2- to 2.5-fold increase in UCP3 protein. Palmitate oxidation was increased in muscle cells incubated under normoglycemic or hyperglycemic conditions, whereas adenoviral green fluorescent protein infection or chronic low doses of the uncoupler dinitrophenol had no effect. Increased UCP3 did not affect glucose oxidation, whereas dinitrophenol and insulin treatments caused increases. Basal oxygen consumption, assessed in situ using self-referencing microelectrodes, was not significantly affected, whereas dinitrophenol caused increases. Mitochondrial membrane potential was decreased by dinitrophenol but was not affected by increased UCP3 expression. Finally, mitochondrial ROS production decreased significantly with increased UCP3 expression. Results are consistent with UCP3 functioning to facilitate fatty acid oxidation and minimize ROS production. As impaired fatty acid metabolism and ROS handling are important precursors in muscular insulin resistance, UCP3 is an important therapeutic target in type 2 diabetes.