Regulation of β cell glucokinase by S-nitrosylation and association with nitric oxide synthase

Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic β cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured β cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic β cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS

Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates

Nutrient-stimulated insulin secretion in mouse islets is critically dependent on intracellular pH

BACKGROUND: Many mechanistic steps underlying nutrient-stimulated insulin secretion (NSIS) are poorly understood. The influence of intracellular pH (pH(i)) on insulin secretion is widely documented, and can be used as an investigative tool. This study demonstrates previously unknown effects of pH(i)-alteration on insulin secretion in mouse islets, which may be utilized to correct defects in insulin secretion. METHODS: Different components of insulin secretion in mouse islets were monitored in the presence and absence of forced changes in pH(i). The parameters measured included time-dependent potentiation of insulin secretion by glucose, and direct insulin secretion by different mitochondrial and non-mitochondrial secretagogues. Islet pH(i )was altered using amiloride, removal of medium Cl(-), and changing medium pH. Resulting changes in islet pH(i )were monitored by confocal microscopy using a pH-sensitive fluorescent indicator. To investigate the underlying mechanisms of the effects of pH(i)-alteration, cellular NAD(P)H levels were measured using two-photon excitation microscopy (TPEM). Data were analyzed using Student's t test. RESULTS: Time-dependent potentiation, a function normally absent in mouse islets, can be unmasked by a forced decrease in pH(i). The optimal range of pH(i )for NSIS is 6.4–6.8. Bringing islet pH(i )to this range enhances insulin secretion by all mitochondrial fuels tested, reverses the inhibition of glucose-stimulated insulin secretion (GSIS) by mitochondrial inhibitors, and is associated with increased levels of cellular NAD(P)H. CONCLUSIONS: Pharmacological alteration of pH(i )is a potential means to correct the secretory defect in non-insulin dependent diabetes mellitus (NIDDM), since forcing islet pH(i )to the optimal range enhances NSIS and induces secretory functions that are normally absent

Disrupting actin filaments enhances glucose-stimulated insulin secretion independent of the cortical actin cytoskeleton

Just under the plasma membrane of most animal cells lies a dense meshwork of actin filaments called the cortical cytoskeleton. In insulin-secreting pancreatic β cells, a long-standing model posits that the cortical actin layer primarily acts to restrict access of insulin granules to the plasma membrane. Here we test this model and find that stimulating β cells with pro-secretory stimuli (glucose and/or KCl) has little impact on the cortical actin layer. Chemical perturbations of actin polymerization, by either disrupting or enhancing filamentation, dramatically enhance glucose-stimulated insulin secretion. Using scanning electron microscopy, we directly visualize the cortical cytoskeleton, allowing us to validate the effect of these filament-disrupting chemicals. We find the state of the cortical actin layer does not correlate with levels of insulin secretion, suggesting filament disruptors act on insulin secretion independently of the cortical cytoskeleton

An orally available compound suppresses glucagon hypersecretion and normalizes hyperglycemia in type 1 diabetes

Suppression of glucagon hypersecretion can normalize hyperglycemia during type 1 diabetes (T1D). Activating erythropoietin-producing human hepatocellular receptor type-A4 (EphA4) on α cells reduced glucagon hypersecretion from dispersed α cells and T1D islets from both human donor and mouse models. We synthesized a high-affinity small molecule agonist for the EphA4 receptor, WCDD301, which showed robust plasma and liver microsome metabolic stability in both mouse and human preparations. In islets and dispersed islet cells from nondiabetic and T1D human donors, WCDD301 reduced glucagon secretion comparable to the natural EphA4 ligand, Ephrin-A5. In diabetic NOD and streptozotocin-treated mice, once-daily oral administration of WCDD301 formulated with a time-release excipient reduced plasma glucagon and normalized blood glucose for more than 3 months. These results suggest that targeting the α cell EphA4 receptor by sustained release of WCDD301 is a promising pharmacologic pathway for normalizing hyperglycemia in patients with T1D

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living β-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs’ properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living β-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions

Scanning electron microscopy of human islet cilia

Human islet primary cilia are vital glucose-regulating organelles whose structure remains uncharacterized. Scanning electron microscopy (SEM) is a useful technique for studying the surface morphology of membrane projections like cilia, but conventional sample preparation does not reveal the submembrane axonemal structure, which holds key implications for ciliary function. To overcome this challenge, we combined SEM with membrane-extraction techniques to examine primary cilia in native human islets. Our data show well-preserved cilia subdomains which demonstrate both expected and unexpected ultrastructural motifs. Morphometric features were quantified when possible, including axonemal length and diameter, microtubule conformations, and chirality. We further describe a ciliary ring, a structure that may be a specialization in human islets. Key findings are correlated with fluorescence microscopy and interpreted in the context of cilia function as a cellular sensor and communications locus in pancreatic islets

When Two Is Better Than One: Elements of Intravital Microscopy

What are the technical underpinnings of two-photon microscopy? What are the advantages of using two-photon microscopy versus conventional confocal microscopy

Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 ± 1.1 μm in length and 0.8 ± 0.2 μm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65 (−/−) mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat (−/−) mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production