Colloidal gold-labeled insulin complex. Characterization and binding to adipocytes

Biologically active insulin gold complex was used as an ultrastructural marker to study insulin binding sites, uptake, and internalization in isolated rat adipocytes. The preparation conditions for monodispersed particles, ca. 16 nm in diameter and loaded with approximately 100 insulin molecules, are reported. The complex is stable for at least six weeks. Single particles or small clusters were scattered across the cell membrane. The distribution of unbound receptors seemed to be independent of the extensive system of pre-existing surface connected vesicles in adipocytes. The uptake of particles took place predominantly via non-coated pinocytotic invaginations; clathrin-coated pits did not seem to be important for this process. Lysosome-like structures contained aggregates of 10-15 particles. These data suggest that insulin gold complex is a useful marker for the specific labeling of insulin binding sites.     

Colloidal gold-labeled insulin complex

Summary Biologically active insulin gold complex was used as an ultrastructural marker to study insulin binding sites, uptake, and internalization in isolated rat adipocytes. The preparation conditions for monodispersed particles, ca. 16 nm in diameter and loaded with approximately 100 insulin molecules, are reported. The complex is stable for at least six weeks. Single particles or small clusters were scattered across the cell membrane. The distribution of unbound receptors seemed to be independent of the extensive system of pre-existing surface connected vesicles in adipocytes. The uptake of particles took place predominantly via non-coated pinocytotic invaginations; clathrin-coated pits did not seem to be important for this process. Lysosome-like structures contained aggregates of 10–15 particles. These data suggest that insulin gold complex is a useful marker for the specific labeling of insulin binding sites.Supported by Deutsche Forschungsgemeinschaft D3 SFB 87     

Lag period for phototropism inPilobolus crystallinus sporangiophores

The lag period for the second positive curvature was examined inPilobolus crystallinus sporangiophores. The lag period for curvature development was 20–30 min at lower fluence rates than 6.32 nmol/m²s but greatly extended at higher fluence rates. When a 20-min symmetrical irradiation with blue light was applied before a 20-min unilateral blue light irradiation, sporangiophores bent as much as those unilaterally and continuously irradiated for 40 min. However, when a 20-min unilateral irradiation was followed by a 20-min symmetrical irradiation, sporangiophores did not show any curvature. That is, the reaction during the first 20 min of the lag period is independent of light direction. This light-direction-independent lag period is considered to be the duration required for adaptation. The lag period for phototropism was also extended when fluence rate was reduced after the start of irradiation. These results suggested that an adaptation process is involved in phototropism ofPilobolus.     

Insulin particle formation in supersaturated aqueous solutions of poly(ethylene glycol)

Protein microspheres are of particular utility in the field of drug delivery. A novel, completely aqueous, process of microsphere fabrication has been devised based on controlled phase separation of protein from water-soluble polymers such as polyethylene glycols. The fabrication process results in the formation of spherical microparticles with narrow particle size distributions. Cooling of preheated human insulin-poly(ethylene glycol)-water solutions results in the facile formation of insulin particles. To map out the supersaturation conditions conducive to particle nucleation and growth, we determined the temperature- and concentration-dependent boundaries of an equilibrium liquid-solid phase separation. The kinetics of formation of microspheres were followed by dynamic and continuous-angle static light scattering techniques. The presence of PEG at a pH that was close to the protein's isoelectric point resulted in rapid nucleation and growth. The time elapsed from the moment of creation of a supersaturated solution and the detection of a solid phase in the system (the induction period, t(ind)) ranged from tens to several hundreds of seconds. The dependence of t(ind) on supersaturation could be described within the framework of classical nucleation theory, with the time needed for the formation of a critical nucleus (size <10 nm) being much longer than the time of the onset of particle growth. The growth was limited by cluster diffusion kinetics. The interfacial energies of the insulin particles were determined to be 3.2-3.4 and 2.2 mJ/m(2) at equilibrium temperatures of 25 and 37 degrees C, respectively. The insulin particles formed as a result of the process were monodisperse and uniformly spherical, in clear distinction to previously reported processes of microcrystalline insulin particle formation.     

Immunocytochemical evidence for the presence of S-100 protein in insulin-containing cells of cultured fetal rat islets

S-100 protein was long considered to be specific to glial and Schwann cells, but was subsequently proved to be present in various organs. In particular, S-100 proteinimmunoreactivity was demonstrated in the parathyroid gland, adenohypophysis and endocrine pancreas. In the present study cultured fetal rat islets were investigated in view of the possible presence of S-100 protein immunoreactivity in their cells. In the initial 5-day period, continuity between islets and ducts could be demonstrated, and the islets appeared to bud from the ducts. During this time, S-100 protein-immunoreactive cells were found in either the budding islets or ducts. The colocalization of S-100 protein and insulin was demonstrated immunocytochemically. In contrast, the newly formed islets from endocrine monolayers did not display S-100 protein immunoreactivity. After this initial period, numerous free-floating islets were observed, but only some of them contained S-100 protein immunoreactivity. S-100 protein-immunoreactive cells had the same distribution as those storing insulin, again suggesting the coexistence of the two peptides. The results suggest that S-100 protein might be involved in the regulation of islet function.     

Coupling of insulin receptors to glucose transport: a temperature-dependent time lag in activation of glucose transport.

We have studied the ability of occupied insulin receptors to activate (or couple to) the glucose transport system in isolated rat adipocytes. Maximal insulin action is seen when only a small proportion (<10%) of the receptors is occupied, and this fraction can be rapidly filled (<5 s) at an insulin concentration of 100 ng/ml. Additionally, control studies show that when the extracellular glucose concentration is tripled, the rate of transport triples within 10 s, indicating that changes in transport activity can be observed nearly instantaneously. Therefore, when cells are exposed to a high insulin concentration (100 ng/ml), any delay in the onset of insulin action beyond this time must be due to the time required for coupling of occupied insulin receptors to the glucose transport system. At 24 °C there is a lag of at least 200 s after insulin addition before a significant stimulation of 2-deoxyglucose transport is seen. The length of this lag phase is temperature dependent, decreasing to 45 s at 37 °C. An Arrhenius plot of the coupling lag is linear, with an activation energy of 25 kcal/mol. After the delay in the onset of initial transport activation the full response appears in a gradual manner, requiring 20 min at 24 °C to attain maximal stimulation. The time required for the full insulin response to appear is also temperature dependent, decreasing to 5 min at 37 °C. Similar results were obtained for the kinetics of insulin activation of 3-O-methyl glucose transport. Thus, the coupling of insulin receptors to the glucose transport system can be divided into two components: an initial absolute time lag followed by a gradual incremental process before the maximal, or full, effect of insulin is achieved. In conclusion, (1) there is an absolute delay in the onset of the insulin's initial action on glucose transport, (2) after an initial delay, activation of transport proceeds in a gradual manner, and (3) the coupling process between insulin receptors and the glucose transport system is temperature dependent and can be described by a linear Arrhenius plot. This suggests that the rate of activation is not limited by membrane fluidity.     

Slow interaction of islet-activating protein with pancreatic islets during primary culture to cause reversal of alpha-adrenergic inhibition of insulin secretion

The manner in which islet-activating protein (IAP), a protein purified from the culture medium of Bordetella pertussis, interacts with the islet B-cell was studied by following the progressive development of IAP-induced reversal of alpha-adrenergic inhibition of insulin release during maintenance of islets in culture with glucose and epinephrine. This action of IAP developed in an exponential manner dependent on its concentration after a true lag period of about 1 h. The lag period was not grossly dependent on the concentration of IAP added but highly dependent on temperature of culture, and was still seen upon adding a second dose of IAP to partially stimulated cells. After 24-h culture significantly more insulin was secreted with IAP at a concentration as low as 1 pg/ml and the half-maximal effect was observed at 0.1 ng/ml. The development of IAP action occurred even in the islets that had been exposed to IAP for only 30 s, but was significantly prevented by anti-IAP serum added before the end of the lag period. IAP was effective in the presence of cycloheximide, an inhibitor of protein synthesis, or of vinblastine or cytochalasin B, microtubular-microfilamentous modifiers. It is suggested that the IAP molecule is rapidly bound to the receptor area of the islet B-cell and then is gradually inserted into the cell membrane before appearance of its action to activate native calcium ionophores. This slow interaction of IAP with the membrane may be responsible for potentiation of insulin secretory and cAMP responses of the cell to various stimuli as well as for reversal of alpha-adrenergic inhibition.     

Effects of streptomycin on plastids in dividing Euglena

Summary The plastids of dividing Euglena cells growing in the light in the presence of streptomycin decreased in length after a lag period of seven generations. The typical structure of the chloroplast was lost after a similar lag period. This loss of structure did not follow a regular pattern. After 11 generations the plastids resembled normal proplastids of dark-grown cells. Initial chlorophyll loss of treated cells was slow, but after 3 generations the rate of loss was about 0.5/generation, indicating a cessation of synthesis and a dilution among the progeny.     

Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase

Summary Malate synthase was investigated by the small-angle X-ray scattering technique in aqueous solution. Measurements extending for several hours revealed a continuous increase of the intensity in the innermost portion of the scattering curve. There is clear evidence that this increase was caused by an X-ray induced aggregation of enzyme particles during the performance of the small-angle X-ray scattering experiment. The monitoring of the aggregation process in situ by means of small-angle X-ray scattering led to a model of the way how the aggregation might proceed. The analysis of the scattering curves of malate synthase taken at various stages of aggregation established the retention of the thickness factor of the native enzyme and the occurrence of one and later on of two cross-section factors. The process of aggregation was also reflected by the increase of extension of the distance distribution function. According to these results, the first step of aggregation might be a linear side-by-side association of the oblate enzyme particles, a process which is followed by a twodimensional aggregation. An aggregation in the third dimension was not observed during the time covered by our experiment. The predominance of aggregation in only one or two dimensions was corroborated by comparison of appropriate theoretical scattering curves with the experimental curves. The theoretical scattering curves for this comparison were obtained by averaging over the properly weighted scattering curves calculated for various species of hypothetical aggregates. The time dependence of the apparent mean radius of gyration was used to compare the aggregation of enzyme samples that were irradiated under different experimental conditions. It turned out that by addition of dithiothreitol to the enzyme solutions as well as in the presence of the substrates (acetyl-CoA, glyoxylate) or of a substrate analogue (pyruvate) or of ethanol the rate of aggregation is reduced. Enzymic activity was found to decrease about exponentially with increasing X-ray dose. The presence of dithiothreitol or of the substrate glyoxylate or of the substrate analogue pyruvate protects the enzyme against X-ray induced inactivation. The substrate acetyl-CoA does not exhibit a comparable protective effect against inactivation. Measurements of enzymic activity and small-angle X-ray scattering on samples, which had been X-irradiated with a defined dose prior to the measurements, established two different series of efficiency for the protection of the enzyme against aggregation (pyruvate > glyoxylate > acetyl-CoA) and inactivation (glyoxylate > pyruvate > $$ " align="middle" border="0"> acetyl-CoA). The results showed that there is no direct relation between the extent of aggregation and the loss of enzymic activity.     

Inhibition of the glucose induced insulin release by somatostatin in the isolated perfused rat pancreas. Action of cyclic AMP, glucagon and glibenclamide.

Insulin release in the perfused isolated rat pancreas was measured after stimulation with 16.5 mM glucose with and without somatostatin (cycle form, 100 ng/ml) in the medium. A complete blockage of the typical biphasic pattern of insulin release ocurred with somatostatin in the medium. Such blockage was abolished when cAMP (2.5 mM) and a 0.5 ml solution of glucagon (1 mg/ml) were continuously perfused for 20-minute periods and for 30-second periods correspondently. It did not take place when glibenclamide (HB-419) was perfused for a 20-minute period at a rate of 10 mug/ml. The results suggest that the adenylcyclase dependent mechanisms of glucose-induced insulin release are involved in the inhibition of the glucose-induced insulin secretion by somatostatin.     

Formation of microparticulate protein powder using a supercritical fluid antisolvent

Gas antisolvent (GAS) expansion of dimethylsulfoxide (DMSO) and N,N-dimethylformamide (DMFA) solutions with supercritical carbon dioxide was used to produce biologically active powders of insulin. Powders with 90% of the particles smaller than 4 mum and 10% smaller than 1 mum were obtained under all conditions tested when the process was operated continuously, with small liquid droplets sprayed into a flowing supercritical continuum. Slow pressurization of the stagnant protein solution resulted in larger particles. In vivo tests on rats revealed no differences between the biological activity of processed and unprocessed insulin, GAS processing of organic solution appears to be a reliable and effective method for the production of dry, biologically active microparticulate powders of peptides and proteins. (c) 1993 John Wiley & Sons, Inc.     

Insulin-like effects of ATP on adipocyte pyruvate dehydrogenase and phosphorylase

Extracellular ATP stimulated adipocyte pyruvate dehydrogenase in a time- and dose-dependent manner with an EC50 of 0.1 mM. The maximal effect was observed at 0.5 mM ATP after a 15-min incubation with a lag period of about 5 min. Depletion of intracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid reduced the effect of ATP by 50% and completely abolished the stimulatory effect of vasopressin on adipocyte pyruvate dehydrogenase but had no effect on the stimulation induced by insulin or adenosine. The effects of insulin and ATP on pyruvate dehydrogenase were glucose-dependent whereas the effect of adenosine was glucose-independent. Furthermore, ATP, like insulin, partially blocked the stimulatory effect of isoproterenol on phosphorylase. Adenosine, at a concentration of 1 mM, did not affect either basal or isoproterenol-stimulated phosphorylase activities. It is concluded that ATP activates adipocyte pyruvate dehydrogenase by at least two separate mechanisms: one is Ca2(+)-dependent and the other is Ca2(+)-independent. However, neither is the result of the formation of adenosine from ATP through hydrolysis.     

Variations of glucose utilization in muscle and adipose tissue of sand rats (Psammomys obesus) during adaptation to laboratory holding

Sand rats, captured in Egypt and fed with a low caloric vegetable diet during adaptation, were investigated before and after 2.5 and 8 weeks diet treatment (30 and 40 kcal/100 g body weight daily). In hexobarbital anaesthesia the sand rats were loaded with 1 g glucose/kg body weight in a single dose intravenously. After a rapid increase the content of glucose in blood remained at a level of about 600 mg glucose/100 ml blood. The insulin immunoreactivity in blood did not change uniformly after application of glucose and remained in a physiologic range. In the islets of Langerhans a degranulation was found during diet treatment. The sensitivity of the epididymal adipose tissue towards insulin in vitro decreased to a nearly complete resistance in the course of diet treatment. A diminution of insulin sensitivity was also found in the m. soleus in vitro. The content of glucose-6-phosphate in the m. semimembranosus was found enhanced after the preparation of the animal. It was found progressively increased up to the five-fold at the end of diet treatment. In the corresponding muscle the glucose distribution volume was increased to about double the extracellular volume. An accumulation of free glucose within the muscle cell must be taken into account. In conclusion the treatment of sand rats with a diabetogenic diet results very quickly in a loss of insulin sensitivity of adipose tissue. The progressively increased stress-mediated accumulation of glucose-6-phosphate and free glucose refers to an inhibition of glucose utilization in the phosphorylation step of glucose in skeletal muscle.     

Wave-induced sediment resuspension in the Öre estuary,northern Sweden

Material transported by the Öre River in northern Sweden is all deposited within the estuary which means that resuspension is necessary for the transport of particles out of the estuary. Wave-induced sediment resuspension in the estuary was studied by monitoring the distribution of suspended particles during a resuspension-redeposition cycle. The particle concentration in the water mass was measured with a light scattering probe, calibrated by comparison with the amount of particles collected on a filter.After a long period with calm weather and a low river input less than 100 tonne of suspended particulate matter was present in the estuary. However, during a period with stormy conditions significant resuspension of sediment particles occurred within the estuary. Two days after the storm approximately 1125 tonne of suspended particulate matter was found in the estuary. Most (61%) of the suspended matter was found in the deepest third of the water column, although up to 17% was present in the top third of the water column. The total load of particulate matter in the water column remained constant until day four after the storm, but a significant redistribution of the particulate matter occurred both in the vertical and horizontal directions. Nine days after the storm, a significant amount of particles (c. 350 tonnes) was still in suspension.     

Effects of insulin on inositol phosphate production in cultured rat hepatocytes

Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li+. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5-5 nM insulin and did not involve changes in [3H]inositol lipid labelling or IP3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18-24 h) or cholera toxin (100 ng/ml for 3-4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li+ was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.     

Collagen fibrillogenesis in vitro: an investigation of the thermal memory effect and of the early events occurring during fibril assembly using dynamic light scattering

Dynamic light scattering has been used to characterize a variety of lathyritic rat skin collagen solutions. The technique was used to monitor the onset of fibril assembly in vitro and to investigate the thermal memory effect. Although the incorporation of thermal memory was demonstrated by reheating the sample and subsequently observing a shortened turbidimetric lag phase, no significant differences between naive solutions and ones exhibiting thermal memory could be detected using photon correlation spectroscopy. This suggests that subtle changes in the state of the collagen molecules rather than extensive changes in the degree of aggregation are responsible for the thermal memory effect. During fibrillogenesis, no large-scale changes in the distribution of monomers or aggregates occur until near the end of the lag phase.     

Assembly of Adenoviruses

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified incomplete particles of adenoviruses type 2 and 3 revealed that core proteins V and VII and capsid proteins VI, VIII, and X were absent in these particles, but they contained polypeptides not present in complete particles. Two types of incomplete particles were observed in the electron microscope, appearing as deoxyribonucleic acid-less particles with single discontinuities in the capsid structure (about 80%), or more amorphous particles resembling hexon aggregates (about 20%). The amount of incomplete and complete particles increased in parallel during the infectious cycle. Detectable amounts were found at 13 h with a maximum rate of synthesis for both particles at 24 h after infection. (3)H-labeled amino acids were incorporated into incomplete particles without a detectable lag period, but the label appeared in complete particles with a 60- to 80-min lag. Early after the pulse in pulse-chase experiments, the radioactivity was higher for incomplete particles than for complete particles and leveled off before the activity of complete particles reached a maximum. In the adenovirus type 2 system, pulse-chase experiments suggested a precursor-product relationship between incomplete and complete particles. After a short pulse, 19 h postinfection, entrance of (3)H-labeled amino acids into the hexon polypeptide of complete particles was delayed for 80 min, but no delay was observed for the labeling of the hexon polypeptide of incomplete particles. The core polypeptides appear in complete particles without a delay, also suggesting that incomplete particles were precursors to complete particles. Incorporation of (3)H-labeled amino acids into the hexon polypeptide of complete and incomplete particles was drastically decreased by inhibition of protein synthesis with emetine. However, the uptake of label into core proteins of complete particles was only decreased to 50% on inhibition of protein synthesis. The results suggest that incomplete particles are intermediates in virus assembly in vivo and that the assembly of capsid polypeptides into incomplete and complete particles is dependent on continuing protein synthesis.     

Degradative processing of internalized insulin in isolated adipocytes

Based on the distribution of 125I-insulin between the cell surface and the cell interior, it was found that insulin rapidly binds (t 1/2 = 0.4 min) to surface receptors at 37 degrees C, and after an initial lag period of about 1 min, accumulates intracellularly until steady state is reached (t 1/2 = 3.5 min). At this time about 40% of the total cell-associated 125I-insulin resides in the cell interior reflecting a dynamic equilibrium between the rate of insulin endocytosis and the rate at which internalized insulin is processed and extruded from cells. Since this percentage decreased to 15% at 16 degrees C, it appears that internalization is more temperative-sensitive than the intracellular processing of insulin. When 125I-insulin was preloaded into the cell interior, it was found that internalized insulin was rapidly released to the medium at 37 degrees C (t 1/2 = 6.5 min) and consisted of both degraded products and intact insulin (as assessed by trichloroacetic acid precipitability and column chromatography). Since 75% of internalized insulin was ultimately degraded, and 25% was released intact, this indicates that degradation is the predominant pathway. To determine when incoming insulin enters a degradative compartment, cells were continually exposed to 125I-insulin and the composition of insulin in the cell interior over time was assessed. After 2 min all endocytosed insulin was intact, between 2-3 min degradation products began accumulating intracellularly, and by 15 min equilibrium was reached with 20% of internalized insulin consisting of degraded products. Degraded insulin was then released from the cell interior within 4-5 min after endocytotic uptake, since this was the earliest time chloroquine was found to inhibit the release of degradation products. Moreover, the final release of degraded insulin was not inhibitable by the energy depleter dinitrophenol. Thus, within the degradative pathway, insulin enters lysosomes by 2.5-3 min and is released to the medium by simple diffusion after an additional 1.5-2 min.     

A continuous process for the production of baculovirus using insect-cell cultures

Summary A continuous culture of insect cells (Spodoptera frugiperda) was used for continuous production of baculovirus (nuclear polyhedrosis virus fromAutographa californica). The system consisted of a cascade of two continuous stirred tank reactors (CSTRs). In CSTR I the insect cells were grown in suspension. This suspension was fed continuously to CSTR II where the virus infection occurred. For a period of about 25 days the average volumetric productivity was about 10⁷ polyhedra (virus particles occluded in protein capsules) and 10⁸ infectious NOVs (non-occluded virus particles) per cm³ effluent. This is equivalent to 25 polyhedra and 250 NOVs per infected cell, respectively. In one case, the percentage of infected cells was 65%, which is close to the theoretical value of 68%. After a run-time of 32 days a decrease of process productivity was observed, probably due to the so-called passage effect, a degeneration of the virus DNA.     

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5°C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5°C on a series of solutions prepared by low-temperature quenching after incubation at 20°C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.