Spinal curvature of young adult females.
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Adolescent↗
Biomedical subjects
S Marshall
Publications and source records attributed to S Marshall.
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.
Adipose Tissue↗
Dual pathways for the intracellular processing of insulin. Relationship between retroendocytosis of intact hormone and the recycling of insulin receptors.
Adipocytes process insulin through either of two pathways: a retroendocytotic pathway that culminates in the release of intact insulin, and a degradative pathway that terminates in the intracellular catabolism and release of degraded ligand. Mechanistically, these pathways were found to differ in several ways. First, temporal differences were found in the rate at which intact and degraded products were extruded. After 125I-insulin was preloaded into the cell interior, intact ligand was completely released during the first 10 min (t 1/2 = 2 min), whereas degraded insulin was released at a much slower rate over 1 h (t 1/2 greater than 8 min). Secondly, it was found that chloroquine profoundly inhibited the insulin degradative pathway, resulting in the intracellular accumulation of intact ligand and a reduction in the release of degraded products. In contrast, however, chloroquine was without effect on the retroendocytotic processing of insulin. Based on the known actions of chloroquine, it appears that retroendocytosis of insulin does not involve vesicular acidification or dissociation of the insulin-receptor complex and that insulin is most likely carried to the cell exterior in the same vesicles (either receptor-bound or free) as those mediating recycling receptors. Interestingly, accumulation of undergraded insulin within chloroquine-treated cells did not result in the release of additional intact ligand, suggesting that once insulin enters the degradative compartment it is committed to catabolism and cannot exit the cell through the retroendocytotic pathway. A third difference was revealed by the finding that extracellular unlabeled insulin (100 ng/ml) markedly accelerated the rate at which preloaded 125I-insulin was released from adipocytes (t 1/2 of 3 min versus 7 min in controls cells). Analysis of the composition of the released products revealed that extracellular insulin rapidly augmented (over 10 min) in a dose-dependent manner (5-200 ng/ml) the amount of insulin released intact (from 25 to 38% of preloaded counts; insulin ED50 = 10 ng/ml). Although extracellular insulin had no effect on the early extrusion of degraded insulin, the release of catabolized products was reduced at later times. The interpretation of these results is that the rate or amount of incoming insulin-receptor complexes can effect a sorting process (prior to bifurcation) such that a proportion of insulin is shunted from the slower degradative pathway to the more rapid retroendocytotic pathway.(ABSTRACT TRUNCATED AT 400 WORDS)
Adipose Tissue↗
Stoichiometric translocation of adipocyte insulin receptors from the cell-surface to the cell-interior. Studies using a novel method to rapidly remove detergent and concentrate soluble receptors.
A rapid one-step method was developed for harvesting and concentrating insulin receptors from solubilized adipocytes, which entails precipitating soluble receptors with polyethylene glycol and resuspending the receptor-containing pellet in a reduced volume of binding buffer. With this procedure 90-100% of receptors were recovered, while 80% of cellular protein was removed, thus resulting in a marked reduction of both ligand and receptor proteases and about a 5-fold purification of the receptor. More importantly, greater than 98% of the Triton X-100 detergent was removed during this procedure so that the reduced receptor affinity observed in solubilized extracts (due to detergent) was restored to normal. Reconstituted receptors exhibited normal binding characteristics similar to those observed for plasma membrane receptors. The general utility of our receptor precipitation-reconstitution method is highlighted by studies on insulin-induced translocation of receptors from the cell-surface to the cell-interior of adipocytes and studies on the assessment of the binding affinity of nascent intracellular receptors. The results of these studies are consistent with the following. 1) Insulin initiates endocytotic uptake of insulin receptors, which then recycle back to the cell-surface. 2) Chloroquine impairs the recycling of internalized receptors while preventing receptor degradation, resulting in the progressive trapping and accumulation of receptors within cells during insulin treatment. 3) Receptor translocation during acute insulin-induced down-regulation is stoichiometric in that receptors lost from the cell-surface can be quantitatively recovered within the cell-interior. 4) In the absence of ligand, these receptors within adipocytes are mainly newly synthesized receptors enroute to the cell-surface, and they possess an affinity similar, if not identical, to mature receptors on the plasma membrane.
Adipose Tissue↗
Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling.
The kinetics of receptor internalization and recycling was directly determined in adipocytes by measuring 125I-insulin binding to total, intracellular, and cell-surface insulin receptors. In the absence of insulin 90% of all receptors were on the cell-surface and 10% were intracellular. Insulin (100 ng/ml) rapidly altered this distribution by translocating surface receptors to the cell-interior through a temperature and energy dependent process. Surface-derived receptors were seen within cells as early as 30 s and accumulated intracellularly at the rate of approximately 20,000/min (t 1/2 = 2.7 min). After 6 min the size of the intracellular receptor pool plateaued (for up to 2 h), with 30% of surface receptors residing within the cell. This plateau was due to the attainment of an equilibrium between receptor uptake and recycling, since removal of insulin (to stop receptor uptake) was followed by both a rapid depletion of intracellular receptors and a a concomitant and stoichiometric reappearance of receptors on the cell-surface. Receptors were efficiently recycled, with little or no net loss observed even after 4 h of insulin treatment; however, recycling could be partially inhibited (approximately 10%) by several agents (e.g. chloroquine and Tris). Tris treatment of adipocytes in the presence of insulin led to 50% loss of surface and total receptors at 2 and 4 h, respectively. Since chloroquine prevented the decrease in total receptors, but not the loss of surface receptors, it appears that Tris impairs recycling by diverting a portion of incoming receptors to a chloroquine-inhibitable degradative site. From these results we conclude that: 1) insulin triggers endocytotic uptake of insulin-receptor complexes; 2) internalized receptors are then rapidly reinserted into the plasma membrane, and the receptors can traverse this recycling pathway within 6 min; 3) prolonged recycling does not normally result in measurable receptor loss, but when receptors are prevented from recycling, they become trapped intracellularly and are shunted to a chloroquine-sensitive degradative pathway; and 4) chloroquine and Tris are only partially effective inhibitors of receptor recycling.
2,4-Dinitrophenol↗
Rupture of prostatic urethral valves via vesicostomy.
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Humans↗
Simple arthroscopic partial meniscectomy associated with anterior cruciate-deficient knees.
Twenty-five patients with combined tears of the meniscus and anterior cruciate ligament in the same knee were evaluated for the results of a simple arthroscopic meniscectomy that preserved the meniscal rim. These patients, classified as "recreational athletes," were analyzed to determine their postoperative functional capabilities. Using the presence or absence of the pivot shift sign as the most important indicator of functional capability, we found that partial meniscectomy was effective in allowing patients to regain a high degree of normal functional ability and in permitting forward motion activities. However, all patients were left with an anterior cruciate-deficient knee that caused laxity in the anterior plane and frequently in the rotatory plane. Rotatory laxity markedly limits activities, and those patients unable to adjust to their instability are considering further surgery.
Adolescent↗
Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes.
To study the regulation of adipose tissue lipoprotein lipase (LPL) in human adipocytes, omental adipose tissue was obtained from healthy subjects and digested in collagenase. The isolated adipocytes thus obtained were suspended in Medium 199 and cultured at 37 degrees C. Cell viability was demonstrated in adipocytes cultured for up to 72 h by constancy of cell number, cell size, trypan-blue exclusion, and specific 125I-insulin binding. In addition, chloroquine induced an increase in cell-associated 125I-insulin at 24, 48, and 72 h after preparation. Thus, isolated adipocytes retained their ability to bind, internalize, and degrade insulin. LPL was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests. A broad range of heparin concentrations produced a prompt release of LPL from a rapidly replenishable pool of cellular activity. When cells were cultured in medium containing 10% fetal bovine serum, there was a marked stimulation of CM and HR. The secretory response to serum (CM) correlated strongly with HR 24 h after preparation (rs = 0.731, P less than 0.001). In addition, HR was found to correlate logarithmically and inversely with body mass index (r = -0.731, P less than 0.001). Insulin, at 400 ng/ml only, increased HR by 36 +/- 10%, an effect simulated by lower concentrations of insulin-like growth factor-1 (IGF1). Thus, LPL is produced and regulated in isolated human adipocytes. The degree of adiposity and serum are important regulators of HR activity, whereas insulin is stimulatory only at a pharmacologic concentration. This effect of insulin may be mediated through the IGF1 receptor. Isolated human adipocytes represent a novel and useful system for the study of LPL and lipid metabolism as well as for other aspects of adipocyte biology.
Adipose Tissue↗
Insulin receptor down-regulation is linked to an insulin-induced postreceptor defect in the glucose transport system in rat adipocytes.
We have examined the relationship between insulin-induced receptor downregulation and the induction of a postreceptor defect in the insulin-stimulated glucose transport system in rat adipocytes, and found that downregulation was linked to the expression of the postreceptor defect. When recycling of insulin receptors was inhibited by 20 mM Tris, insulin pretreatment (100 ng/ml) for 4 h at 37 degrees C induced both net loss (65%) of cell-surface receptors and a 63% decrease in maximal insulin responsiveness. In contrast, when cells were treated with insulin alone for 4 h at 37 degrees C so that receptors could recycle, or treated at 16 degrees C with Tris plus insulin to inhibit receptor internalization, neither receptor downregulation nor a postreceptor defect was observed. Induction of the postreceptor defect was specific for insulin under conditions when downregulation would occur, since treatment of cells with Tris and the insulin mimicker spermine did not result in receptor loss or the postreceptor defect. Other experiments revealed that receptor downregulation occurred first without loss of insulin responsiveness, but, once the postreceptor defect appeared, its severity was correlated to the degree of further receptor loss, as a function of insulin dose and exposure time. Tris (20 mM) alone acutely decreased maximally stimulated glucose transport rates slightly (22%), but this effect was rapidly reversible after Tris removal and could not have been directly responsible for the lasting and profound postreceptor defect seen after pretreatment with insulin plus Tris. Taken together, these data suggest that insulin-induced receptor loss is linked to the induction of the postreceptor defect. The postreceptor defect was due to an inability to maximally increase the maximum velocity of glucose transport. Furthermore, the expression of the postreceptor defect depended upon the extent to which the glucose transport system was allowed to deactivate; maintaining the glucose transport system in an activated state prevented its expression. Thus, the mechanism could involve rapid inactivation or sequestration of glucose transporters during deactivation such that they become refractory to the subsequent stimulatory effects of insulin. In conclusion, (a) insulin does not acutely induce a postreceptor defect in the glucose transport system of adipocytes without loss of cell-surface insulin receptors; (b) the defect in stimulated glucose transport has been induced distal to the insulin receptor via a mechanism linked to receptor loss; and (c) the postreceptor lesion is due to decreased number of intrinsic activity of glucose transporters on the cell-surface in the presence of a maximally effective insulin concentration. These data suggest that insulin receptor downregulation and postreceptor defects in insulin action, which frequently co-exist both in vivo and in vitro, may be linked mechanistically.
Adipose Tissue↗
Etiology, diagnosis, and treatment of recovery room bleeding/hemorrhage.
A successful outcome for the patient who hemorrhages after surgery is based on the recognition of bleeding, the specific diagnosis of bleeding sites and disorders, and the management of bleeding by control measures and replacement of blood lost (Table III). The best preventative measures include careful preoperative assessment of historical and physical findings along with appropriate laboratory tests.
Blood Coagulation Factors↗
Alternative routes and methods of insulin delivery.
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Diabetes Mellitus↗
Primary culture of isolated adipocytes. A new model to study insulin receptor regulation and insulin action.
The mechanism by which insulin regulates cell surface insulin receptors was examined in primary cultured rat adipocytes. When cells were incubated in insulin-free medium, specific 125I-insulin binding progressively increased over 3-4 days followed by a plateau of binding. Insulin prevented up-regulation at low doses (1 ng/ml), while higher doses (5-25 ng/ml) resulted in a net loss of surface receptors. A lag period of 4-6 h preceded insulin-induced changes in receptor number, and such a lag was seen prior to the inhibitory effect of insulin on the insertion of nascent receptors into the plasma membrane. Regulation of surface receptors continued after the removal of insulin, consistent with the idea that insulin generates a signal which can sustain receptor regulation in the absence of ligand. Thus, a 1-h insulin pulse (100 ng/ml) was sufficient to block up-regulation, whereas longer exposure times (4-12 h) produced a net loss of surface receptors. When cells were exposed to insulin for a fixed time (5 h), subsequent receptor loss was insulin dose-dependent. Thus, the net number of cell surface insulin receptors is determined by both insulin concentration and the duration of insulin exposure. Time course studies after a 12-h insulin pulse revealed a progressive loss of surface receptors for up to 36 h. At later times receptor number returned toward control values, thus demonstrating that triggering of receptor regulation is reversible. To determine whether insulin-induced down-regulation was mediated by receptor loss, or by receptor translocation to an intracellular site, we measured receptor distribution (cell surface and intracellular pools) in control and 72-h insulin-treated adipocytes. These data revealed that down-regulation was mediated by a net loss of receptors rather than by receptor redistribution. An early biological response of adipocytes to insulin is rapid endocytosis of insulin-receptor complexes, and we found that insulin plays an important role in regulating this endocytotic uptake rate. Thus, compared to freshly isolated cells, adipocytes cultured for 72 h in insulin-free medium had an enhanced ability to internalize both insulin and insulin receptors, whereas cells chronically treated with insulin endocytosed insulin-receptor complexes at a much slower rate. These data are interpreted as an insulin-mediated change in one of the cellular responses of adipocytes to the biological action of insulin.
Adipose Tissue↗
Spermidine cytotoxicity in vitro: effect of serum and oxygen tension.
Plasma amine oxidase activities (benzylamine oxidase and spermine oxidase) were determined in the sera of a number of species of various ages. Benzylamine oxidase (BZO) activity, measured spectrophotometrically, was present in bovine, equine, and ovine species examined. Generally its activity in serum increased with the age of the animal. Spermine oxidase activity (SPO) was estimated by a bioassay of in vitro toxicity and did not necessarily correlate with BZO. Cytotoxicity in the presence of spermidine was found only in the sera of the ruminant species examined. Serum activity tended to rise with animal age; however, great variability was found in perinatal bovine sera. The 50% lethal dose (LD50) of spermidine in the presence of 5% serum and 4 X 10(4) NS1 cells/ml was in the micromolar range. Aminoguanidine, a known inhibitor of SPO, could prevent the cytotoxic effects of exogenously added spermidine in vitro. In contrast, raising the ambient oxygen tension in the incubation environment to 95% lowered the LD50 dose of spermidine required for cytotoxicity. The results suggest that a cell line of hematogenous origin is susceptible to the cytotoxic effects of the products of oxidative deamination of spermidine by SPO, an enzyme present in perinatal bovine sera, and that these cytotoxic effects are potentiated in the presence of an oxygen-enriched environment in vitro.
Aging↗
Tyrosinase activity and abundance in Cloudman melanoma cells.
Rabbit anti-tyrosinase antibodies were used to study the abundance, processing, and degradation of tyrosinase in murine (Cloudman) melanoma cells. The polyclonal antibodies precipitated low-molecular-weight (68,000 and 70,000) and high-molecular-weight (78,000 and 80,000) tyrosinases that had a precursor-product relationship. Cells with high basal tyrosinase activity had high levels of newly synthesized tyrosinase. Cells with low tyrosinase activity synthesized less tyrosinase and degraded the enzyme at a faster rate than cells with high tyrosinase activity. Melanotropin (melanocyte stimulating hormone), dibutyryl cyclic adenosine monophosphate, and isobutylmethylxanthine caused an increase in the abundance of newly synthesized tyrosinase that was directly proportional to the increase in enzyme activity. This enzyme was not a phosphoprotein. Other changes in the culture conditions that increased the level of tyrosinase activity increased the abundance of newly synthesized enzyme. It is thus concluded that the level of tyrosinase activity in Cloudman melanoma cells is a direct reflection of the abundance of enzyme protein.
1-Methyl-3-isobutylxanthine↗
Significance of transient elevation of PAP levels after TUR.
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Acid Phosphatase↗
Modification of allograft immunogenicity in perinatal islets isolated and purified in vitro.
Perinatal rat islets of Langerhans, isolated and cultured in vitro, were examined following long-term allotransplantation across a major histocompatibility barrier in nonimmunosuppressed recipients. Islets were isolated to varying degrees of purity without the use of collagenase digestion. Newborn bovine serum was a component of the incubation medium and the atmosphere during culture was air: 5% CO2. Islets transplanted without rigorous purification were fully rejected by 14 days posttransplantation. However, if islets were maintained in subculture, permitting their subsequent meticulous purification, no evidence of rejection was observed after 45 days at the kidney subcapsular site. Grafts consisted of morphologically intact islets. The three major endocrine cell types of the islet were identified by immunocytochemical localization of insulin, glucagon, and somatostatin. These results demonstrate that perinatal islets can exhibit altered immunogenicity, as evidenced by prolonged allograft survival, when isolated and purified by the nonenzymic in vitro method.
Animals↗
Insulin regulation of lipoprotein lipase in cultured isolated rat adipocytes.
The cellular regulation of adipose tissue lipoprotein lipase by insulin was investigated using cultured isolated rat adipocytes. Evidence for sustained cell viability over 3 days included stability of specific [125I]insulin binding and adipocyte number. Lipoprotein lipase was measured in three functional compartments: 1) enzyme activity secreted into the culture medium, 2) activity releasable from cell suspensions by heparin, and 3) activity extractable from cells (after maximal heparin release) in deoxycholate and detergent. One day after preparation, these activities stabilized and were 1.3 +/- 0.2, 1.4 +/- 0.2, and 7.7 +/- 0.9 neq/10(6) cells X min, respectively (n = 24, mean +/- SEM). Insulin, added the day after preparation, produced a dose-dependent (1-400 ng/ml) increase in lipoprotein lipase releasable from cells by heparin at 2, 4, and 24 h. Insulin also increased intracellular enzyme measured as deoxycholate-detergent-solubilized activity extracted from previously heparin-released cells. However, insulin-mediated increases in culture medium enzyme only occurred subsequent to cellular effects. All insulin-mediated effects were prevented by cycloheximide (1 microgram/ml). Thus, insulin increased two cellular pools of adipocyte lipoprotein lipase in a dose-dependent manner, but had no direct effect on enzyme secretion. Overall, cultured isolated rat adipocytes appear to be a valuable system for the study of lipoprotein lipase regulation at the level of the adipocyte.
Adipose Tissue↗
Separate intracellular pathways for insulin receptor recycling and insulin degradation in isolated rat adipocytes.
To gain insight into the sequence of events that follow endocytotic uptake of insulin receptor complexes, we have examined the interrelationship between the degradative pathway of the insulin ligand and the recycling pathway of the insulin receptor. Tris(hydroxymethyl)aminomethane and other nonamphoteric amines were found to selectively impair insulin receptor recycling while leaving the insulin-degradative pathway intact. In contrast, low concentrations of the lysosomotropic agent chloroquine markedly inhibited intracellular insulin degradation but had little or no affect on the recycling of internalized receptors. Thus, we conclude: (1) that insulin dissociates from its receptor after endocytotic uptake and both receptor and ligand follow a separate intracellular pathway; and (2) that receptor recycling and insulin degradation can be selectively inhibited by Tris and chloroquine, respectively, highlighting the potential usefulness of these agents as intracellular probes in the study of receptor-ligand metabolism.
Adipose Tissue↗