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G A Rutter

Publications and source records attributed to G A Rutter.

At least 55 records · Page 3Linked to original sources
PubMed1998

Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase.

We have prepared recombinant cDNAs encoding chimaeras between human preproinsulin (sp.B.C.A., for B-, Connecting- and A-peptides) and a thermostable mutant of green fluorescent protein (GFPS65T,V163A, GFP*). The subcellular localization of the expressed chimaeras was monitored in living insulin-secreting INS-1 beta-cells by laser scanning confocal microscopy. When GFP* was fused at the immediate N-terminus of the B-chain (sp.[GFP*].B.C.A.myc) two distinct patterns of fluorescence were apparent. In 1530/1740 cells examined, fluorescence was confined to a reticular, exclusively extranuclear structure, and closely co-localized with the endoplasmic reticulum marker, calreticulin. However, 210/1740 (12.1%) of cells displayed punctate fluorescence, which partially co-localized with the trans-Golgi network marker, TGN 38, and with the dense core secretory granule marker, phogrin. Since secretion of GFP* fluorescence into the medium could not readily be measured, we prepared a chimaera in which firefly luciferase was fused at the C-terminus of proinsulin (sp.B.C.A.myc.[Luc]). This chimaera displayed a distribution closely similar to that of sp.[GFP*].B.C.A. myc, but with a lower proportion (15/310, 4.8%) of the cells showing clear punctate distribution. At substimulatory glucose concentrations (3 mM) secretion of sp.B.C.A.myc.[Luc] could not be detected (rate of release into the medium identical with that of the cytosolic Renilla reniformis luciferase), indicating that the chimaera did not enter the constitutive secretory pathway. However, elevated (30 mM) glucose stimulated the release of the sp.B.C.A.myc. [Luc] luciferase chimaera, without a detectable effect on R. reniformis luciferase release. These data suggest that fusion of insulin, and the much larger photoproteins GFP* and luciferase, leads predominantly to misfolding and retention in the endoplasmic reticulum. However, the properly folded chimaeras are apparently still correctly targeted to the regulated, rather than the constitutive, secretory pathway. These chimaeras should therefore be valuable tools to monitor the exocytosis of insulin in real time.

Cell Line↗
PubMed1998

A phogrin-aequorin chimaera to image free Ca2+ in the vicinity of secretory granules.

Microdomains of high Ca2+ concentration ([Ca2+]) may be critical to the control of intracellular processes such as secretion and metabolism without compromising other cell functions. To explore changes in [Ca2+] in the outer mantle (< 30 nm deep) that surrounds the surface of dense-core secretory granules, we have designed a recombinant chimaera between the granule protein phogrin and aequorin. When expressed in populations of insulin-secreting MIN6 or phaeochromocytoma PC12 cells, the chimaera was targeted to secretory granules as expected. The recombinant protein reported a similar [Ca2+] at the granule surface to that in the bulk cytosol, measured with untargeted aequorin. This was the case both at rest (-Ca2+- = 80-120 nM) and after stimulation with agents that provoke Ca2+ entry or Ca2+ mobilization from intracellular pools, and during activated secretion. Thus depolarization of MIN6 cell populations with high K+ increased [Ca2+] both in the bulk cytosol and close to the granules to approx. 4 microM, with near-identical kinetics of increase and recovery. Similarly, stimulation of PC12 cells with ATP provoked an increase in -Ca2+- in either domain to 1.3 microM. These data argue that, in MIN6 and PC12 neuroendocrine cells (i) significant mobilization of Ca2+ from most secretory granules probably does not occur during activated Ca2+ influx or mobilization of internal Ca2+ stores, and (ii) agonist-stimulated Ca2+-dependent secretion can occur without development of a large gradient of [Ca2+] between the surface of most secretory vesicles and the rest of the cytosol.

Adenosine Triphosphate↗
PubMed1998

Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes.

OBJECTIVE: Mitochondrial [Ca2+] ([Ca2+]m) rises in parallel with cytosolic [Ca2+] ([Ca2+]c) following ATP-depletion rigor contracture induced by hypoxia in isolated cardiomyocytes. We investigated the pathways involved in the hypoxia induced changes in [Ca2+]m by using known inhibitors of mitochondrial Ca2+ transport, namely ruthenium red, an inhibitor of the Ca2+ uniporter (the normal influx route) and clonazepam, an inhibitor of Na+/Ca2+ exchange, (the normal efflux route). METHODS: [Ca2+]m was determined from indo-1/am loaded rat myocytes where the cytosolic fluorescence signal had been quenched by superfusion with Mn2+. [Ca2+]c was measured by loading myocytes with indo-1 pentapotassium salt during the isolation procedure. Cells were placed in a specially developed chamber for induction of hypoxia and reoxygenated 40 min after rigor development. RESULTS: 50% of control cells hypercontracted upon reoxygenation; this correlated with a [Ca2+]m or [Ca2+]c higher than approximately 350 nM at the end of rigor. Clonazepam completely abolished the rigor-induced rise in [Ca2+]m but not [Ca2+]c. On reoxygenation [Ca2+]m increased over the first 5 min and remained elevated whereas [Ca2+]c fell. In the presence of ruthenium red a dramatic increase in [Ca2+]m occurred 5-10 min after rigor development (the indo-1 fluorescence signal was saturated); [Ca2+]c also increased but to a lesser extent. On reoxygenation, [Ca2+]m fell rapidly even though cells hypercontracted and [Ca2+]c remained elevated. CONCLUSIONS: During hypoxia following rigor development Ca2+ uptake into mitochondria occurs largely via the Na+/Ca2+ exchanger rather than the Ca2+ uniporter whereas on reoxygenation the transporters resume their normal directionality.

Animals↗
PubMed1998

Real-time imaging of gene expression in single living cells.

Recent advances in reporter gene technologies are now allowing us to image gene transcription at the single cell level, using either fluorescence or luminescence microscopy. Here, the basis of these techniques is outlined and their advantages and disadvantages in various biological systems are discussed.

Animals↗
PubMed1997

Upstream stimulatory factor-2 (USF2) activity is required for glucose stimulation of L-pyruvate kinase promoter activity in single living islet beta-cells.

Elevated glucose concentrations stimulate L-pyruvate kinase (L-PK) gene transcription in liver and islet beta-cells. A glucose response element termed the L4 box (two noncanonical E-boxes located -165 and -154 base pairs upstream of the transcriptional start point) has previously been defined within the proximal promoter region of the gene. However, the identity of the transacting factor(s) which binds to this site remains unclear. We have used photon counting digital imaging of firefly luciferase activity to monitor promoter activity continuously in single living islet beta and derived INS-1 cells, and to analyze the molecular basis of the regulation by glucose. L-PK promoter activity, normalized to cytomegalovirus promoter activity using the distinct Renilla reniformis luciferase, was >/=6-fold higher in cells cultured at 16 mM glucose or above compared with cells cultured at 3 mM glucose. Microinjection of antibodies against the ubiquitous transcription factor USF2 inhibited L-PK promoter activity in beta- and INS-1 cells incubated at 30 mM glucose by 71-87%. Anti-USF2 antibodies had a much smaller effect on promoter activity in INS-1 cells cultured at 3 mM glucose, and on the activity of a modified promoter construct lacking an L4 box. These data support the view that glucose enhances L-PK gene transcription in beta-cells by modifying the transactivational capacity of USF2 bound to the upstream L4 box.

Animals↗
PubMed1996

Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.

Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.

Aequorin↗
PubMed1996

The hormonal regulation of pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex has a central role in the regulation of mammalian metabolism as it represents the point-of-no-return in the utilization of carbohydrate. This article summarizes our studies into how signalling systems initiated by hormones binding to cell surface receptors can reach the pyruvate dehydrogenase system which is located within the inner mitochondrial membrane. One class of hormones which activate pyruvate dehydrogenase are those that increase cytoplasmic Ca2+. A wide range of studies on isolated enzymes, separated mitochondria and intact cell preparations have shown that the activation is due to the stimulation of pyruvate dehydrogenase phosphatase. Two other intramitochondrial dehydrogenases which regulate the citrate acid cycle are activated in parallel and this is an important means of balancing the supply of ATP to increasing cell demand. Insulin is also able to activate pyruvate dehydrogenase, but this is restricted to fat and other cells capable of lipogenesis. Insulin acts by stimulating pyruvate dehydrogenase phosphatase, but the activation does not involve alterations in Ca2+. The signalling pathway involved has not been established, but it appears to be quite distinct from those involved in many other actions of insulin.

Acetyl-CoA Carboxylase↗
PubMed1995

Involvement of MAP kinase in insulin signalling revealed by non-invasive imaging of luciferase gene expression in single living cells.

BACKGROUND: Studies of the mechanisms by which signals are transmitted from receptor tyrosine kinases would be facilitated by a way of monitoring events at the single-cell level. We have explored how luciferase imaging can be used to examine the role of specific signalling pathways in insulin-stimulated gene expression. The analysis of luciferase expression in single cells has previously been hampered by the insensitivity of existing methodologies and the lack of a way of monitoring quantitatively, and independently, more than one promoter within the same cell. We have developed a technique for examining the dynamics of insulin-stimulated AP-1-dependent transcription in single living cells, and have explored the signalling pathway involved. RESULTS: Luciferase and aequorin gene expression were examined in single living cells with a high-sensitivity photon-counting camera. The technique involved the comicroinjection of luciferase- and aequorin-based reporter plasmids directly into the cell nucleus, and the subsequent analysis of luminescence in the presence of luciferin and coelenterazine, respectively. The method is quantitative and allows insulin-stimulated gene expression to be monitored in real time. We found that insulin promoted a substantial increase in the expression of a luciferase gene under the control of the AP-1-binding site from the collagenase gene promoter. Aequorin expression, under the control of a cytomegalovirus promoter, was unaffected by insulin. The effect of insulin on luciferase expression was specifically blocked by overexpression of either the mitogen-activated protein (MAP) kinase phosphatase CL100, or the dominant-negative mutant MAP kinase kinase, MEKS217/221A. CONCLUSIONS: Microinjection coupled with luciferase imaging allows hormone-regulated gene expression from relatively weak promoters to be monitored in single living cells. We have used this method to demonstrate that MAP kinase plays a central role in the ability of insulin to stimulate AP-1-dependent gene transcription.

Aequorin↗
PubMed1994

Ca2+ stores in insulin-secreting cells: lack of effect of cADP ribose.

Ca2+ stores were examined in several insulin secreting cell types by measuring uptake and release of Ca2+ by permeabilised cells. In pancreatic islet cells or INS-1 cells, < 20% of the ATP-dependent, thapsigargin-sensitive Ca2+ pool could be released by saturating concentrations of inositol (1,4,5)P3 (InsP3). InsP3 released > 60% of the thapsigargin-sensitive Ca2+ pool in RINm5F cells. The total Ca2+ content of the thapsigargin-sensitive pool was similar in each of these cell types. Neither cADP ribose (cADPR; 1 microM) nor caffeine (10 mM) caused significant Ca2+ release from any of the permeabilised insulin-secreting cell preparations. ATP elicited similar increases in intracellular Ca2+ concentration ([Ca2+]i) in single, living INS-1 and RINm5F cells, and similar fold increases in InsP3 levels in cell populations. The Ca2+ ATPase inhibitor thapsigargin, added after ATP, caused smaller [Ca2+]i increases in RINm5F than in INS-1 cells. This is consistent with the presence of a smaller InsP3-sensitive Ca2+ pool in living INS-1 cells. The data indicate that InsP3 receptors are present in only a small subfraction of the Ca2+ ATPase-containing Ca2+ stores in INS-1 and pancreatic beta-cells, and that cADP ribose/caffeine-sensitive Ca(2+)-induced Ca2+ release channels may be entirely absent from this endocrine cell type.

Adenosine Diphosphate Ribose↗
PubMed1993

Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion.

The effects of stimulated Ca2+ influx on cytosolic ([Ca2+]c) or intramitochondrial free Ca2+ ([Ca2+]m) were examined in the new pancreatic beta-cell line, INS-1. [Ca2+]c was monitored by video imaging of single fura-2-loaded INS-1 cells, or in populations of cells transfected with non-targeted (cytosolic) aequorin. [Ca2+]m was measured after transfection with aequorin targeted to the mitochondria by fusion of the gene in frame with the signal peptide of cytochrome c oxidase subunit VIII. Two physiological stimuli of native beta-cells, glucose and ATP, raised [Ca2+]c in INS-1 cells largely by stimulating Ca2+ influx. Thus, glucose (20 mM) induced repetitive transient increases in [Ca2+]c (0.42 min-1, mean amplitude 229 nM above 102 nM basal). These transients were largely due to periodic stimulation of Ca2+ influx through voltage-sensitive Ca2+ channels, since they could be rapidly and reversibly blocked by chelation of external Ca2+, by addition of the hyperpolarizing agent diazoxide, or with the Ca2+ channel blocker SR 7037. ATP, by contrast, caused single transient [Ca2+]c increases, to about 300 nM above basal levels, which could be inhibited by > 90% upon external Ca2+ chelation. Challenge of aequorin-transfected cells with ATP increased [Ca2+]m to 4 microM or above, an effect blocked by EGTA. Furthermore, plasma membrane depolarization with high K+, used as a glucose surrogate to mimic, in a synchronized fashion, the influx-induced Ca2+ transients observed at the single-cell level, also increased [Ca2+]m to > 4 microM. Similar increases in [Ca2+]m were also measured in other aequorin-transfected insulin-secreting cells, RINm5F, during mobilization of internal Ca2+ with carbachol. In contrast, glucose-induced changes in [Ca2+]m were below the level of detection in INS-1 cell populations, consistent with the asynchrony of the [Ca2+]c transients induced by this nutrient at the single-cell level, and the consequent small average [Ca2+]c rise. These data are in line with the view that stimulated Ca2+ influx into excitable cells raises [Ca2+]m as efficiently as internal Ca2+ mobilization in nonexcitable cells. In the case of INS-1 and pancreatic beta-cells, this may be important both to enhance oxidative metabolism, hence fueling the secretory process, and also to maintain the production of metabolic signaling molecules.

Adenosine Triphosphate↗
PubMed1992

Regulation of mitochondrial glycerol-phosphate dehydrogenase by Ca2+ within electropermeabilized insulin-secreting cells (INS-1).

(1) A new insulin-secreting cell line (INS-1; Asfari et al. (1992) Endocrinology 130, 167-178) has been used to study the regulation by Ca2+ of mitochondrial FAD-linked glycerol-phosphate dehydrogenase (FAD-GPDH) in situ. (2) Enzyme activity was examined on-line in electropermeabilized cells by a new, sensitive, assay. This involved the reduction of the artificial electron acceptor, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), monitored by the quenching of the fluorescence of rhodamine-18. Using this approach, similar total levels of FAD-GPDH activity (nmol/min per 10(6) cells) were measured in INS-1 cells (1.35 +/- 0.22) and isolated rat islet cells (1.63 +/- 0.02) (3) Ca2+ ions markedly activated the enzyme, lowering the apparent Km-value for added DL-glycerophosphate from 8.8 +/- 1.4 mM to 1.0 +/- 0.1 mM. Ca2+ had no effect on the apparent Vmax. The enzyme displayed cooperative kinetics with respect to DL-glycerophosphate (Hill coefficient of 2.0 +/- 0.2 and 1.6 +/- 0.2 in the absence and presence respectively of Ca2+). Half-maximal effects of Ca2+ were observed in the range 30-130 nM, depending on the concentration of glycerol phosphate. (4) Enzyme activity was weakly (30%) inhibited by diazoxide, but not by the diabetogenic drug, streptozotocin. (5) The data indicate that INS-1 cells represent an excellent model for studying the rôle of FAD-GPDH in the control of insulin secretion.

Animals↗
PubMed1992

Regulation of pyruvate dehydrogenase by insulin and polyamines within electropermeabilized fat-cells and isolated mitochondria.

1. Regulation of the mammalian pyruvate dehydrogenase (PDH) complex by insulin and polyamines has been examined by using electropermeabilized rat epididymal fat-cells and isolated mitochondria. The complex could be regulated within the permeabilized cells not only by insulin, but also by certain low-M(r) species, including Ca2+ and the polyamine spermidine. 2. Both spermine and spermidine increased the level of active dephosphorylated PDH (PDHa) in isolated adipose-tissue mitochondria 2-3-fold, with half-maximal effects at 0.9 mM and 1.7 mM respectively. By contrast, PDH activity in rat heart mitochondria was essentially insensitive to the effects of these polyamines. 3. The effects on PDH activity of incubation of adipose-tissue mitochondria with spermine persisted through re-isolation and re-incubation of the mitochondria in the absence of the polyamine. 4. No evidence was found of any increase in the concentration of spermine associated with purified mitochondrial fractions prepared from insulin-treated tissue. 5. Overall, the data provide further evidence against a role for polyamines in the rapid stimulation of PDH by insulin, but suggest that polyamines may be important in mediating longer-term changes in the activity of the complex.

Adipose Tissue↗
PubMed1992

Effects of insulin and guanosine 5'-[gamma-thio]triphosphate on fatty acid synthesis and lipolysis within electropermeabilized fat-cells.

1. Exposure to electric fields ('electroporation') has been used to investigate the mechanism of the action of insulin on the regulation of fatty acid synthesis and lipolysis in isolated rat fat-cells. 2. Exposure of the cells to electric fields (six pulses at 2 or 3 kV/cm) permitted the uptake of a number of low-Mr molecules normally excluded from cells, including sucrose (Mr 342), EDTA (Mr 394) and propidium iodide (Mr 668). At least 90% of the cells were found to be permeable to these species. 3. Insulin stimulated the synthesis of fatty acids in electroporated (2 kV/cm) cells to a similar extent (0.68 +/- 0.19 to 6.7 +/- 0.7 micrograms-atoms of glucose carbon/h per g, or 3.3 +/- 0.6 to 11.2 +/- 1.6 micrograms-atoms of H2O hydrogen/h per g) to that observed in non-electroporated cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]; 0.5 mM) also stimulated fatty acid synthesis (2-fold) and flux of glucose carbon into triacylglycerol glycerol (3-fold) in these cells, but had no effect on these parameters in non-electroporated cells. 4. Lipolysis in the electroporated cells was stimulated by isoprenaline and also by GTP[S], but only the effects of isoprenaline were inhibited by insulin. 5. Exposure to a higher field strength (3 kV/cm) gave results qualitatively similar to those described above, although the effects of insulin and isoprenaline were diminished. 6. These studies provide evidence against a role for changes in Ca2+, and probably also Na+, K+ or Mg2+, in insulin action on fat-cells, but may support a role for GTP-binding proteins.

Adipose Tissue↗
PubMed1991

Effects of protein phosphatase inhibitors on the regulation of insulin-sensitive enzymes within rat epididymal fat-pads and cells.

1. The effects of the protein phosphatase inhibitors okadaic acid and microcystin LR on the regulation by insulin of pyruvate dehydrogenase and acetyl-CoA carboxylase have been studied in rat epididymal fat-pads and isolated cells. These inhibitors both completely blocked the phosphatase activity (against phosphorylase a) present in extracts of epididymal fat-pads, with half-maximal effects in the nanomolar range. 2. Okadaic acid treatment of pads and cells lowered the activity of acetyl-CoA carboxylase assayed in tissue extracts, both before and after treatment of the extracts with the activator, citrate. Further, okadaic acid treatment abolished the 2-3-fold difference in activity observed between extracts from control and insulin-treated tissues, assayed without prior treatment with citrate. 3. Incubation of pads with [32P]Pi, sufficient to label the intracellular pool of ATP, demonstrated that okadaic acid increased the overall phosphorylation of acetyl-CoA carboxylase on a number of distinct sites, as judged by two-dimensional mapping of tryptic peptides. These included the 'I-peptide' [Brownsey & Denton (1982) Biochem. J. 202, 77-86], the phosphorylation of which may be associated with the stimulation of the activity of the enzyme by insulin, as well as inhibitory phosphorylation sites. 4. Incubation with 1 microM-okadaic acid had no effect on the basal level of active pyruvate dehydrogenase apparent after tissue extraction, but abolished the 2-3-fold increase in this parameter which was elicited by insulin in the absence of okadaic acid. However, okadaic acid treatment did not affect the persistent increase in active pyruvate dehydrogenase levels which was apparent in mitochondria subsequently isolated from insulin-treated pads and re-incubated with an oxidizable substrate. It is concluded that the effects of okadaic acid are exerted through changes in metabolite concentrations rather than some direct action on the signalling pathway whereby insulin stimulates pyruvate dehydrogenase. 5. Microcystin LR did not mimic the effects of okadaic acid on intact cells and pads described above.

Acetyl-CoA Carboxylase↗
PubMed1990

Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes.

1. The concentration of free Mg2+ ([Mg2+]m) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent Kd values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above Kd values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+]m to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaCl had no significant effect on the fall in [Mg2+], whereas 1 microM-nigericin blocked, and 0.3 microM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.

Animals↗