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Biomedical subjects

L Jarett

Publications and source records attributed to L Jarett.

At least 127 records · Page 7Linked to original sources
PubMed1980

Differences in organizational structure of insulin receptor on rat adipocyte and liver plasma membranes: role of disulfide bonds.

Binding of 125I-labeled insulin to rat liver and adipocyte plasma membranes has been investigated after treatment of the membranes with agents that modify disulfide bonds or sulfhydryl groups. Dithiothreitol, a disulfide-reducing agent, produced a bimodal response in adipocyte plasma membranes with dose-dependent increases in binding occurring over the range of 0-1 mM dithiothreitol; 5 mM dithiothreitol produced decreased binding. Insulin binding reached its maximal increase at 1 mM and was 3 times control values. Scatchard analysis of the 1 mM dithiothreitol effect revealed a straight line plot indicative of one class of sites with a Ka of 1.0 x 10(8) M-1 which is intermediate between the two Kas obtained from the curvilinear Scatchard plot of control membranes. There was a 20-fold increase in the number of intermediate-affinity receptors compared to high-affinity receptors. The increased 125I-labeled insulin binding after dithiothreitol treatment was reversed by oxidized glutathione in a dose-dependent manner. Interposition of treatment with N-ethylmaleimide, an alkylating agent, prevented oxidized glutathione from reversing the dithiothreitol effect. Reduced glutathione produced the same effect as dithiothreitol. Liver plasma membranes treated with up to 1 mM dithiothreitol exhibited a maximum increase in insulin binding of 20% compared to control. Dithiothreitol at 5 mM decreased insulin binding below that of control membranes. The results indicate that the dithiothreitol effect on insulin binding to adipocyte plasma membranes is due to disruption of disulfide bonds, and that the structural organization of the insulin receptor on the plasma membranes is different for liver and for adipose tissue. The data imply that the insulin receptors on the plasma membrane of adipocytes possess at least two functionally distinct subclasses of disulfide bond but liver insulin receptors do not.

Adipose Tissue↗
PubMed1980

Surface structure changes of rat adipocytes during lipolysis stimulated by various lipolytic agents. A scanning electron microscopic study.

A qualitative and quantitative electron microscopic study was performed on rat adipocytes during stimulation of lipolysis by various agents. Scanning electron microscopy of control cells revealed a spherical cell with a textured glycocalyx surface exhibiting small irregular projections. Globular surface evaginations or protrusions measuring 8-18 muM in diameter were seen on cell hemispheres, and there was an average of one protrusion for every two hemispheres examined. Distribution analysis showed that 60 percent of the hemispheres had no protrusions, and 25, 10, and 5 percent of the hemispheres had one, two or three protrusions, respectively. Thin-section and freeze- fracture electron microscopy of the protrusions showed a small triglyceride droplet surrounded by a thin cytoplasmic rim that was continuous with the main cytoplasmic matrix. The glycocalyx coating and plasma membrane extended from the cell surface onto, and over, the protrusion. Scanning microscopy of cells stimulated by lipolytic agents, including epinephrine, adrenocorticotropic hormone, theophylline, and dibutyryl cyclic AMP, revealed a dose-dependent increase in the number of protrusions per cell hemisphere. Maximal concentrations of lipolytic hormones cuase an average 2.5-fold increase in the number of protrusions per hemisphere without changing the average size of the protrusions. Only 40 percent of the stimulated cell hemispheres exhibited no protrusions; over 15 percent of the cells contained three or more; and a number of the protrusions were multilobulate. Insulin prevented the increase in the number of protrusions and the change in distribution caused by the lipolytic hormones but did not prevent the increase caused by theophylline and dibutryl cyclic AMP. The data suggest that the protrusions are a structural feature of the cell and may be related to the lypolytic pathway. These observations may help explain some of the discrepant biochemical data relating to hormonal stimulation of lipolysis.

Adipose Tissue↗
PubMed1980

The effect of epinephrine on calcium handling by adipocyte plasma membranes, endoplasmic reticulum, and mitochonrdia.

The effect of epinephrine treatment of adipocytes upon calcium handling by subsequently isolated plasma membranes, endoplasmic reticulum, and mitochondria has been examined and compared to the previously reported effects of insulin upon these parameters. Epinephrine (0.1 micrograms/ml) treatment of adipocytes resulted in: 1) a 19% increase in calcium binding to plasma membranes at calcium concentrations (2 and 250 microM) which reflect binding to the high and low affinity calcium-binding sites, respectively; 2) small but significant decreases in the maximum rate (-7.4%) and the Km (-12.6%) for calcium uptake by the endoplasmic reticulum without changes in the steady state capacity or the rate of efflux from the endoplasmic reticulum; and 3) no change in the stable pool of calcium in the mitochondria. The epinephrine-induced increase in calcium binding by the plasma membranes is identical to the effects of insulin previously reported, suggesting that this alteration may be involved in the similar membrane effects of these two hormones. In contrast, the effects of epinephrine on endoplasmic reticulum and mitochondrial calcium are strikingly different from the effects of insulin, suggesting that these parameters may be related to the vastly different effects that these hormones have on intracellular metabolic events.

Adipose Tissue↗
PubMed1980

A comparison of basal and insulin-stimulated glucose transport in rat adipocyte plasma membranes.

Specific D-glucose transport in plasma membranes prepared from control and insulin-treated rat adipocytes was measured using a recently developed dual isotope ([3H]-D-glucose and [14C]-L-glucose), rapid filtration assay which allowed measurements of initial rates at 1 s and 37 degrees. Plasma membranes from insulin-treated adipocytes showed an increase in glucose transport compared with control cells. Saturation kinetic data revealed that the plasma membranes from insulin-treated and control cells had the same Km (26 mM) for glucose transport, whereas insulin treatment increased the Vmax from 4433 pmol/mg protein/s to 9465 pmol/mg proteins/s. Arrhenius plots showed no difference in the energy of activation between control and insulin-stimulated glucose transport states. The optimum pH of both control and insulin-stimulated glucose transport was 7.4. Lower or higher pHs progressively decreased both control and insulin-stimulated glucose transport proportionately. Calcium in the transport assay media did not affect basal or insulin-stimulated glucose transport. However, omission of calcium from the adipocyte incubation media significantly lowered the insulin stimulation by 24% while basal levels were not significantly affected. Insulin specifically bound to the plasma membrane was carried through the fractionation procedure, but removal of this insulin did not alter the stimulated glucose transport. Glucose transport by plasma membranes from control or insulin-treated adipocytes was equally (percentage) inhibited by N-ethylmaleimide, dithiothreitol, reduced glutathione, or cytochalasin B. No inhibition of control or insulin-stimulated transport was seen with cytochalasin D or oxidized glutathione. The data presented are consistent with insulin causing the formation of new transport sites similar to the existing basal sites.

Adipose Tissue↗
PubMed1980

Lysosomal degradation of receptor-bound 125I-labeled insulin by rat adipocytes: its characterization and dissociation from the short-term biologic effects of insulin.

In this study we used chloroquine to characterize the interalization and lysosomal degradation of receptor-bound 125I-insulin by rat adipocytes and to determine the role of lysosomal processing of insulin in the short-term biologic effects of the hormone. Chloroquine inhibited the degradation of 125I-insulin bound to adipocytes by both association and disslociation experiments. In the former experiments, chloroquine caused a time- and concentration-dependent increase in specifically bound insulin owing to an increase in intact insulin and a decrease in degradation products, as determined by trichloroacetic acid precipitability and gel chromatographic analysis of material extracted from the cells. In the dissociation experiments, 50 microM chloroquine decreased the rate of degradation by two third, as reflected in the release of degradation to or degraded by isolated plasma membranes, on the degradation of 125I-insulin by proteases in the incubation medium, or on the endocytotic uptake of receptor-bound insulin. Quantitative electron miroscopy, using monomeric ferritin-insulin, showed 50 microM chloroquine doubled the number of lysosomal structures containing ferritin. These findings are consistent with an inhibition by chloroquine of lysosomal degradation of internalized receptor-bound insulin. Chloroquine, at these same concentrations, had no effect on the ability of insulin to stimulate glucose transport and oxidation or to inhibit epinephrine-stimulated lipolysis. In these studies, we show that lysosomal degradation of internalized receptor-bound insulin is not necessary for insulin to cause short-term biologic effects in the adipocyte.

Adipose Tissue↗
PubMed1980

Isolation from rat adipocytes of a chemical mediator for insulin activation of pyruvate dehydrogenase.

Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid-stable material which, when isolated from intact adipocytes by heat extraction and subsequent Sephadex G25 chromatography, yielded a single active fraction that stimulated mitochondrial pyruvate dehydrogenase by activating the phosphatase and not by altering the kinase activity. Phosphatase activation was demonstrated by the ability of the active material to increase pyruvate dehydrogenase activity in the absence of ATP and by the ability of NaF, a phosphatase inhibitor, to this stimulation. Involvement of the kinase in this activation mechanism was eliminated by the fact that, in the presence of ATP, (1) NaF completely blocked the stimulation of pyruvate dehydrogenase by the active fraction, and (2) the stimulation of pyruvate dehydrogenase by dichloroacetic acid, a kinase inhibitor, was additive to the stimulation caused by the active fraction. This active fraction may contain an intracellular chemical mediator or second messenger for insulin.

Adipose Tissue↗
PubMed1979

Pyruvate dehydrogenase activation in adipocyte mitochondria by an insulin-generated mediator from muscle.

Material in a chromatographic fraction from an extract of insulin-treated muscle stimulated pyruvate dehydrogenase activity in addipocyte mitochondria. This action was similar to insulin's activation of the enzyme in a plasma membrane-mitochondria mixture. Neither the chromatographic fraction nor insulin required adenosine triphosphate or magnesium ion (Mg2+), suggesting that both agents acted through a calcium-sensitive phosphatase. This fraction may contain a chemical mediator of insulin action.

Adenosine Triphosphate↗
PubMed1979

A kinetic analysis of D-glucose transport by adipocyte plasma membranes.

The measurement of the initial rate of glucose uptake is of critical importance for the kinetic analysis of the effects of temperature, ions, or hormones on the uptake process. Presented here is a highly reproducible double isotope assay system which for the first time allowed the measurement of the initial rates of glucose uptake into isolated adipocyte plasma membrane vesicles by having time points as early as 1/2 s. Measurement of the initial rates of uptake at 1 s allowed the calculation of a Km of 9.0 mM and a Vmax of 3209 pmol/mg of protein/s for specific D-glucose transport (D-glucose minus L-glucose). The time course of specific D-glucose uptake is extremely rapid, reaching an equilibrium value in approximately 20 s with a half-time of 4 s. This uptake process is markedly inhibited by cytochalasin B. The similarity of these findings to those for intact adipocytes indicates that the plasma membrane preparation and the assay conditions used have retained the structural and functional integrity of the glucose transport system from the intact cell.

Adipose Tissue↗
PubMed1979

Effect of cytochalasin B and D on groups of insulin receptors and on insulin action in rat adipocytes. Possible evidence for a structural relationship of the insulin receptor to the glucose transport system.

The possible physiological importance of the groups of insulin receptors on rat adipocytes and the relationship of these groups to insulin action were investigated. The effect of cytochalasin B and D on biological actions of insulin was measured and compared with the effect of these agents on the ultrastructural distribution of groups of insulin receptors. Cytochalasin B had no effect on epinephrine-stimulated lipolysis, insulin inhibition of epinephrine-stimulated lipolysis, or insulin stimulation of protein synthesis. Cytochalasin B, over a concentration range of 50 nM to 5 muM, progressively inhibited the basal glucose transport system, as measured by glucose oxidation, 2-deoxyglucose transport, and 3-O-methylglucose transport. Insulin was capable of fully stimulating remaining basal transport at submaximal concentrations of cytochalasin B. Insulin pretreatment of adipocytes partially protected the glucose transport system from inhibition by cytochalasin B. Cytochalasin B markedly altered the distribution pattern of insulin receptors, which caused an increase in the number of single receptor molecules by decreasing the number of larger groups. A significant correlation (r = 0.964; P < 0.001) was found between the percent increase in single receptors and the percent decrease in glucose transport. Ferritin-insulin pretreatment of adipocytes prevented disruption of the groups of insulin receptors by cytochalasin B. Cytochalasin D had no effect on the biological actions of insulin or on the groups of insulin receptors. These data suggest that the ability of insulin to affect adipocyte metabolism is independent of the hormone occupying adjacent, grouped receptor sites. The marked contrast in effects of cytochalasin B and D on groups of insulin receptors and glucose transport suggests that the microfilament system is not involved in insulin action or in holding the groups of insulin receptors together, as both agents are known disrupters of microfilaments and inhibitors of actin gelation. The correlation between the effects of cytochalasin B on insulin receptor distribution and glucose transport leads to the speculation that the glycoprotein molecules containing the insulin receptor are functionally linked with the glucose transport system.

Adipose Tissue↗
PubMed1979

Demonstration and partial characterization of insulin receptors in human platelets.

Recently, evidence has been reported to suggest that human platelets like several other circulating blood cells may bind insulin. To examine whether human platelets contain specific insulin receptors, washed human platelets suspended in Hepes buffer were incubated at 24 degrees C with 125I-insulin in the presence and absence of unlabeled insulin and specific insulin binding was determined. Insulin binding by platelets increased progressively with time of incubation to reach a maximum at 3 h and was proportional to the number of platelets in the incubation mixture. Maximum insulin binding was observed at pH 8. Insulin degradation by platelets as assessed by TCA precipitability and reincubation studies was minimal. Scatchard analysis of the binding data and dissociation studies revealed evidence of negative cooperativity of the platelet insulin receptor. A high affinity dissociation constant of approximately equal to 3 X 10(9) M-1 was determined and the concentration of platelet insulin receptors was estimated as 25 binding sites/micron2 platelet surface area. Binding of 125I-insulin by platelets was inhibited by unlabeled porcine insulin and to a lesser extent by catfish insulin and porcine proinsulin but not by glucagon, prolactin, growth hormone, and thrombin. The findings indicate that human platelets contain specific insulin receptors. The significance of the platelet insulin receptor, particularly with respect to altered platelet function in diabetes mellitus, remains to be determined.

Blood Platelets↗
PubMed1978

A high-performance liquid chromatography method for hemoglobin A1c.

Hemoglobin A1c (HbA1c) is a glycosylated derivative of hemoglobin and is one of a family of derivatives whose concentrations are elevated in patients with diabetes mellitus. Published methods for the measurement of HbA1c are relatively tedious and require modest amounts of blood. A high-performance liquid chromatographic (HPLC) method for the determination of HbA1c is presented. The method is rapid (20 minutes), precise (coefficient of variation of 5-10 per cent), uses small amounts of sample (3 microliter.), can be automated. A sample preparation technique using filtration was developed that shortened and simplified preparation of venous blood and allowed use of capillary samples. HbA1c was measured by this method in three age-stratified groups of controls and a group of insulin-requiring juvenile diabetics. There was clear separation of HbA1c values between all normals (5.9 +/- 1.3, 5.6 +/- 0.7, 7.1 +/- 0.9 per cent) and the diabetics (12.1 +/- 2.4 per cent). Use of this method can facilitate large-scale clinical investigations and permit biochemical investigations of the metabolism and formation of hemoglobin A1c where small sample sizes are necessary.

Adolescent↗