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D W Piston

Publications and source records attributed to D W Piston.

At least 19 recordsLinked to original sources
PubMed2001

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps.

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.

3-O-Methylglucose↗
PubMed2001

Substrate-induced nuclear export and peripheral compartmentalization of hepatic glucokinase correlates with glycogen deposition.

Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopy and quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60 min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.

Active Transport, Cell Nucleus↗
PubMed2000

Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells.

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be approximately 30 microM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be approximately 7 microM. Likewise, the NAD(P)H increase of the mitochondrial domain is approximately 60 microM and is delayed with respect to the change in cytoplasmic NAD(P)H by approximately 20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.

Animals↗
PubMed2000

Photobleaching in two-photon excitation microscopy.

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.

Biophysical Phenomena↗
PubMed1999

Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion.

The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between beta cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gklox). Analysis of AdenoCre-treated islets indicated that the gklox allele in approximately 30% of islet cells was converted to a nonexpressing variant (gkdel). This resulted in a heterogeneous population of beta cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet beta cells from gklox/lox mice. In AdenoCre-infected islets, approximately one-third of the beta cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at approximately 5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an KcatS0.5 of approximately 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.

Adenoviridae↗
PubMed1999

A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins.

We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.

Bacterial Proteins↗
PubMed1999

Imaging living cells and tissues by two-photon excitation microscopy.

Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Since two-photon excitation occurs only at the focal point of the microscope, it inherently provides three-dimensional resolution. This localization of excitation also minimizes photobleaching and photodamage, which are the ultimate limiting factors in imaging living cells. Furthermore, no pinhole is required to attain three-dimensional discrimination, so the efficiency of fluorescence collection is increased. These advantages allow experiments on thick living samples that would not be possible with other imaging techniques. The cost and complexity of the lasers required for two-photon excitation microscopy have limited its use, but appropriate turn-key lasers have now been introduced, and their cost should decrease. Finally, the recent introduction of commercial two-photon excitation laser-scanning microscope systems allows a much larger group of researchers access to this state-of-the-art methodology.

Animals↗
PubMed1999

Two-color GFP expression system for C. elegans.

We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.

Amino Acid Substitution↗
PubMed1998

Quantitative imaging of TATA-binding protein in living yeast cells.

We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) x TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP x TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP x TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real-time quantitation in living cells and subcellular localization information. Time-lapse confocal imaging of GFP x TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0.7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP x TBP, we suggest that intracellular levels of TBP are near rate-limiting for growth and viability.

Cell Nucleus↗
PubMed1998

Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.

Fluorescence↗
PubMed1998

Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation.

The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm; > 90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; approximately 35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

3T3 Cells↗
PubMed1998

Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer.

The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin fluorescence. Polarized fluorescence measurements of HPLC-purified band 3 oligomers indicate that eosin homotransfer increases progressively with increasing species size. This shows that homotransfer also occurs between labeled band 3 dimers as well as within the dimers, making fluorescence anisotropy measurements sensitive to band 3 self-association. Treatment of ghost membranes with either Zn2+ or melittin, agents that cluster band 3, significantly decreases the anisotropy as a result of the increased homotransfer within the band 3 clusters. By comparison with the anisotropy of species of known oligomeric state, the anisotropy of erythrocyte ghost membranes at 37 degrees C is consistent with dimeric and/or tetrameric band 3, and does not require postulation of a fraction of large clusters. Proteolytic removal of the cytoplasmic domain of band 3, which significantly increases the rotational mobility of the transmembrane domain, does not affect its oligomeric state, as reported by eosin homotransfer. These results support a model in which interaction with the membrane skeleton restricts the mobility of band 3 without significantly altering its self-association state.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

Choosing objective lenses: the importance of numerical aperture and magnification in digital optical microscopy.

Microscopic images are characterized by a number of microscope-specific parameters--numerical aperture (NA), magnification (M), and resolution (R)--and by parameters that also depend on the specimen--for example, contrast, signal-to-noise ratio, dynamic range, and integration time. In this article, issues associated with the microscope-specific parameters NA, M, and R are discussed with respect to both widefield and laser scanning confocal microscopies. Although most of the discussion points apply to optical microscopy in general, the main application considered is fluorescence microscopy.

Lenses↗
PubMed1997

Cell-specific expression and regulation of a glucokinase gene locus transgene.

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Animals↗