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Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 307 records · Page 17Linked to original sources
PubMed1984

Characterization of the serum from a patient with insulin resistance and hypoglycemia. Evidence for multiple populations of insulin receptor antibodies with different receptor binding and insulin-mimicking activities.

The serum from a patient with lupus nephritis, insulin resistance, and hypoglycemia was studied. This serum both inhibits the binding of 125I-insulin to its receptor and has insulin-like activity on fat cells (see refs. 1 and 2). The IgG fraction from this patient's serum one-half maximally inhibited 125I-insulin binding to IM-9 cells at 1 microM, but did not markedly inhibit 125I-monoclonal antibody binding even at concentrations as high as 4 microM. The IgG was then subjected to affinity chromatography on a protein A-Sepharose column. Four protein peaks were eluted from this column by a step pH gradient from 5.5 to 2.3. Three of the four peaks inhibited 125I-insulin binding to its receptors, but none was more potent than the unfractionated IgG itself. One IgG peak, however, was able to inhibit 125I-monoclonal antibody binding at tenfold lower concentrations than the unfractionated IgG. When the ability of the four IgG fractions to stimulate 2-deoxy[3H]-D-glucose transport in rat adipocytes was studied, two fractions showed stimulatory activity. Compared with unfractionated IgG, one had a weak ability to inhibit 125I-insulin binding, but tenfold more potency to mimic insulin action. The other had a strong ability to inhibit 125I-insulin binding but less potency to mimic insulin action. These studies indicate, therefore, that the serum contains multiple populations of antibodies to the insulin receptor, or portions of the plasma membrane adjacent to the receptor, which have different biologic effects.

Adipose Tissue↗
PubMed1984

Degradation of insulin-like growth factors I and II by a human insulin degrading enzyme.

A human insulin degrading enzyme purified from IM-9 lymphocytes was tested for its ability to degrade insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II). Degradation of these molecules was assessed by trichloroacetic acid precipitation, binding to specific receptors and chromatography on Sephadex G-50. All three techniques indicated that the enzyme readily degraded IGF-II and slightly degraded IGF-I. The IGF-II degrading activity chromatofocused with the insulin degrading activity and was absorbed by specific antibodies to the insulin degrading enzyme. These studies indicate, therefore, that a human insulin degrading enzyme can degrade IGF-II and, to a lesser extent, IGF-I.

Chemical Phenomena↗
PubMed1983

Monocrotaline pyrrole-induced pulmonary hypertension in fawn-hooded rats with platelet storage pool deficiency: 5-hydroxytryptamine uptake by isolated, perfused lungs.

Platelets are believed to be involved in the development of monocrotaline pyrrole (MCTP)-induced pulmonary hypertension. To help identify the role of the platelet, the cardiopulmonary toxicity of MCTP was examined in fawn-hooded (FH) rats, a strain with a platelet function defect. Both Sprague-Dawley (SD) and FH rats developed right ventricular hypertrophy and increased lung weights and exhibited decreased biogenic amine removal by isolated, perfused lung preparations after MCTP treatment. The responses of the FH rats were not significantly different from those of the SD rats, suggesting that platelet uptake and release of 5-hydroxytryptamine (5HT) are not the platelet functions involved in MCTP-induced pulmonary hypertension. The FH rats had an interesting strain-related difference from SD rats; isolated lungs from FH rats removed and metabolized a greater proportion of perfused 5HT than the SD rats.

Animals↗
PubMed1983

Preferential degradation of the beta subunit of purified insulin receptor. Effect on insulin binding and protein kinase activities of the receptor.

Collagenase preparations (a mixture of enzymes including collagenase, clostripain, and a casein-degrading protease) degraded the beta subunit (Mr = 95,000) of the purified insulin receptor into fragments of Mr less than 15,000, without degrading the alpha subunit. The resulting beta-digested insulin receptor preparations were found to bind insulin as well as control insulin receptor, as assessed by either cross-linking of 125I-insulin to the digested receptor or by separating insulin bound to receptor from free insulin by high performance liquid chromatography. Moreover, the beta-digested insulin receptor preparations were still precipitated by a monoclonal antibody directed against the insulin-binding site. In contrast, the beta-digested insulin receptor lacked protein kinase activity since it no longer phosphorylated either itself, or an exogenous substrate, calf thymus histone. These results support the identification of the beta subunit of the insulin receptor as a protein kinase.

Cell Line↗
PubMed1983

Aggregation of platelets from monocrotaline pyrrole-treated rats.

Aggregation responses of platelet-rich plasma (PRP) from monocrotaline pyrrole (MCTP)-treated rats were examined to help elucidate the role of platelets in MCTP-induced pulmonary hypertension. PRP from rats treated one or four days earlier with MCTP (5 mg/kg, i.v.) or vehicle exhibited the same slope and maximum aggregation to ADP (1 X 10(-5) M, 2 X 10(-6) M, 1 X 10(-6) M), 74 micrograms/ml dog collagen, or 500 micrograms/ml arachidonic acid. PRP collected 7 days after MCTP treatment had a 15% decrease in both slope and maximum aggregation to 1 X 10(-5) M ADP and a 25% decrease in response to 2 X 10(-6) M ADP relative to the control group. Fourteen days after MCTP treatment, the responses to all of the aggregating agents were decreased 18-71% from control values. These results indicate that MCTP treatment alters the responses of PRP to aggregating agents.

Adenosine Diphosphate↗
PubMed1983

Regulation of the insulin receptor by a monoclonal anti-receptor antibody. Evidence that receptor down regulation can be independent of insulin action.

In the present study, we investigated the ability of a monoclonal antibody to the insulin receptor to regulate the expression of the insulin receptor of IM-9 lymphocytes. Previously, this antibody was shown to be a competitive antagonist of insulin action on severe metabolic functions. In the present study, we report that preincubation of IM-9 cells with the monoclonal antibody caused a dose- and time-dependent decrease in the subsequent ability of these cells to bind 125I-insulin, a phenomenon termed down regulation. The antibody was approximately 100 times more potent than insulin at down regulating the receptor. In contrast, the antibody was 5 times less potent than insulin in competing for binding to insulin receptors and dissociated 4 times more rapidly than insulin from IM-9 cells. Three lines of evidence suggested that the mechanism of down regulation by the antibody was the same as the one used by insulin. First, both agents caused a rapid initial decrease in insulin binding to cells followed by a slower, gradual decrease in binding. Second, the down regulation caused by both was reversible, and this reversibility required new protein synthesis. Third, the antibody, like insulin, accelerated receptor degradation. Since the antibody does not mimic the other effects of insulin on metabolic processes, these results suggest that the mechanism of insulin receptor down regulation is different from the mechanism of insulin action on other cellular functions.

Antibodies, Monoclonal↗
PubMed1983

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist.

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.

Antibodies↗
PubMed1983

Insulin receptor: evidence that it is a protein kinase.

Highly purified preparations of insulin receptor catalyzed the phosphorylation of the 95,000-dalton subunit of the insulin receptor. This subunit of the insulin receptor was also labeled with [alpha-32P]8-azidoadenosine 5'-triphosphate, a photoaffinity label for adenosine triphosphate binding sites. The identity of the 95,000-dalton band was confirmed in both cases by precipitation with a monoclonal antibody to the insulin receptor. These results suggest that the insulin receptor is itself a protein kinase.

Adenosine Triphosphate↗
PubMed1983

Insulin-cholera toxin binding unit conjugate: a hybrid molecule with insulin biological activity and cholera toxin binding specificity.

The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin-CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20-fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin-CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin-like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.

Amino Acids↗
PubMed1983

The influence of flow on the metabolism of perfused benzo[a]pyrene by isolated rat lung.

In order to investigate the influence of flow and, thus, substrate delivery, on the ability of lung to metabolize foreign compounds, the disappearance of circulating [3H]benzo[a]pyrene ([3H]B[a]P) and the appearance of B[a]P metabolites was monitored in isolated rat lungs from control and 3-methylcholanthrene (3-MC) pretreated rats perfused at low (10 ml/min) and high (45 ml/min) flows. Increasing the flow or 3-MC pretreatment hastened the disappearance of B[a]P from the perfusion medium reservoir and increased the rate of appearance of total metabolites. However, these manipulations affected the appearance of individual metabolites in the medium in different ways. For example, in lungs from control rats the rate of appearance of 7,8-dihydrodiol (7,8-dihydroxy-7,8-dihydro-B[a]P) (7,8-DHD) in the perfusion medium was markedly increased by increasing flow while that of B[a]P-1,6-quinone was minimally affected. In addition, increasing flow increased the concentration of some B[a]P metabolites, such as 4,5-dihydrodiol (4,5-dihydroxy-4,5-dihydro-B[a]P) (4,5-DHD) in the lung tissue of control rats at the end of the perfusion period, but did not effect much change in the concentration of these metabolites in lungs from 3-MC-pretreated rats. The results show that flow, as well as 3-MC pretreatment, may alter the rate at which metabolism of foreign compounds occurs and the temporal profile of metabolites produced by the intact lung.

Animals↗
PubMed1983

Pulmonary hypertension and ECG changes from monocrotaline pyrrole in the rat.

Chemically synthesized monocrotaline pyrrole (MCTP) was administered to adult male rats at a dose of 5 mg/kg in the tail vein. Controls received an equivalent volume of dimethylformamide vehicle. Rats were killed at 3, 5, 7, 10, and 14 days after treatment. Bronchopulmonary lavage fluid lactate dehydrogenase activity and lung weight were significantly elevated at 4 and 7 days, respectively, after MCTP, indicating that pulmonary damage had occurred. White blood cell count was elevated 7 days after treatment. Mean pulmonary arterial pressure was also first elevated in treated (22 +/- 3 mmHg) compared with control (16 +/- 1 mmHg) animals 7 days after treatment. Right ventricle-to-left ventricle plus septum weight ratios were significantly increased in treated (0.429 +/- 0.015) vs. control (0.320 +/- 0.015) animals 14 days after treatment. Development of right heart enlargement correlated with a shift in the QRS complex mean electrical axis in the frontal plane of the electrocardiogram. These results indicate that MCTP produces effects similar to that caused by monocrotaline, that pulmonary arterial pressure increases from control levels between 5 and 7 days after treatment, and that measurement of mean electrical axis of the electrocardiogram may be a useful, noninvasive method to monitor MCTP-induced cardiac changes in vivo.

Animals↗
PubMed1983

Interactions of a monoclonal antibody to the insulin receptor with receptors for insulin-like growth factors.

A monoclonal antibody to the human insulin receptor was tested for its ability to inhibit the binding of 125I-insulin-like growth factor I (IGF-I) and 125I-insulin-like growth factor II (IGF-II) to their receptors in human placenta membranes and cultured human IM-9 lymphocytes. In both placenta membranes and IM-9 cells, the antibody progressively inhibited the binding of 125I-IGF-I to its receptor with a potency that was 300-fold less than its ability to inhibit the binding of 125I-insulin to its own receptor. In contrast, in human placenta membranes, this antibody inhibited the binding of 125I-IGF-II to its receptor only slightly. These studies indicate, therefore, that this monoclonal antibody binds preferentially to the insulin receptor but also crossreacts to a lesser extent with the IGF-I receptor.

Antibodies, Monoclonal↗
PubMed1983

Insulin-ricin B chain conjugate has enhanced biological activity in insulin-insensitive cells.

Insulin-ricin B chain conjugate, a hybrid molecule consisting of insulin covalently linked to the binding chain of ricin, was tested for insulin-like biological activity in HTC and H35 rat hepatoma cells, rat adipocytes, rat thymocytes, and human fibroblasts. In H35 cells and adipocytes, cells that have abundant insulin receptors and are very sensitive to insulin (ED50 of 30 pM and 50 pM, respectively), the conjugate had 5% the biological activity of native insulin (ED50 of 500 pM and 1000 pM, respectively). Since the insulin portion of the conjugate has 5% the potency of native insulin in binding to the insulin receptor, these observations suggested that (in these cells) the conjugate was acting via the insulin receptor. Moreover lactose and galactose, potent inhibitors of ricin binding to its receptor, had no effect on the action of the conjugate on H35 cells. In contrast, in thymocytes, HTC cells, and fibroblasts cells that have relatively few insulin receptors and require high concentrations of insulin to elicit biological actions (ED50 of 10 nM, 20 nM, and 1 nM, respectively), the conjugate had more biological activity than was predicted on the basis of its ability to bind to the insulin receptor. In addition, in these three insulin-insensitive cell types, the activity of the conjugate was inhibited by either lactose or galactose. Thus, these observations indicate that in cells which are relatively insensitive to insulin, the biological effects of the conjugate require the interaction of the ricin B chain moiety with the ricin receptor. In addition, in fibroblasts, the activity of the conjugate was inhibited by the addition of a monoclonal antibody to the insulin receptor which inhibits the response of fibroblasts to insulin. These data suggest, therefore, that in insulin-insensitive cells the binding of the ricin B chain moiety to its receptor enhances the interaction of the insulin portion of the conjugate with the insulin receptor.

Adipose Tissue↗
PubMed1983

Clearance of benzo(a)pyrene by isolated rat liver and lung: alterations in perfusion and metabolic capacity.

The ability of isolated rat livers and lungs to clear circulating benzo(a)pyrene was examined under conditions of altered perfusion and pretreatment with 3-methylcholanthrene (3-MC). In isolated organs from control rats, pulmonary benzo(a)pyrene clearance was low (approximately 1 ml/min) and independent of flow between 10 and 45 ml/min, whereas hepatic clearance was greater and flow dependent, increasing from 3.2 +/- 0.2 ml/min at a flow of 7 ml/min to 13.2 +/- 0.7 ml/min at a flow of 20 ml/min. 3-MC pretreatment increased markedly the arylhydrocarbon hydroxylase activity of broken cell preparations from both organs. However, clearance of benzo(a)pyrene was not increased in isolated livers by 3-MC pretreatment. On the other hand, clearance did increase and became flow dependent in isolated lungs of 3-MC-pretreated rats. In fact, when organs from these animals were perfused at flows occurring in vivo, hepatic and pulmonary clearance were about equal. These results indicate that flow and metabolic capacity are important determinants of both hepatic and pulmonary clearance of xenobiotic compounds and suggest that alterations in either factor may affect the relative roles of these organs in the total body disposition of xenobiotic compounds.

Animals↗
PubMed1983

Total body clearance of circulating benzo(a)pyrene in conscious rats: effect of pretreatment with 3-methylcholanthrene and the role of liver and lung.

The metabolic clearance of benzo(a)pyrene [B(a)P] was studied in conscious control and 3-methylcholanthrene (3-MC)-pretreated rats. The roles of liver and lung in total body B(a)P clearance were estimated in vivo in 3-MC-treated rats. During a 5-hr period, blood B(a)P concentration decreased rapidly in a biphasic manner. 3-MC pretreatment did not significantly affect the rate constants of B(a)P elimination, but the apparent volume of distribution increased, resulting in an increase in total body B(a)P clearance from 15 +/- 1 to 48 +/- 6 ml/min. Five hours after injection, B(a)P concentrations in most organs were lower in 3-MC-pretreated rats; however, lung B(a)P concentration was greater. In the 3-MC-pretreated rats, first-pass extraction of B(a)P by lung and liver was determined from comparison of areas under arterial blood B(a)P concentration vs. time curves after i.a., i.v. and intrahepatic portal venous administration. Liver had a first-pass extraction of 20% whereas that by lung was 33%. Organ B(a)P concentrations were correlated with the route of B(a)P administration. The results of this study suggest that 1) 3-MC pretreatment increases the apparent volume of B(a)P distribution in vivo, 2) liver and lung of 3-MC-pretreated rats are about equal in their ability to extract circulating B(a)P in vivo and 3) 3-MC pretreatment enhances total body B(a)P clearance by increasing extrahepatic B(a)P elimination.

Animals↗
PubMed1983

The prediction of benzo[a]pyrene clearance by rat liver and lung from enzyme kinetic data.

The metabolic clearance of circulating benzo[a]pyrene (B[a]P) by liver and lung of control and 3-methylcholanthrene (3MC)-pretreated rats was predicted according to the perfusion-limited model from apparent enzyme kinetic constants determined in microsomal incubations. These predictions were tested in isolated organs perfused at normal organ flow. From microsomal incubations the apparent enzyme kinetic constants of B[a]P metabolism were determined. The apparent Km of liver microsomes was decreased 100 times by pretreatment with 3MC, while the Km of lung microsomes remained at about 0.2 microM. Maximal velocity of B[a]P metabolism was much greater in microsomes from liver than in those from lung of both control and 3MC-pretreated rats. Liver was found to have a far greater capacity for B[a]P metabolism (intrinsic free clearance) than lung. However, this large disparity was not evident in the predicted clearances. Perfused organs had B[a]P clearances very close to those predicted from the model. At normal (in vivo) organ flows, control rat lung had a clearance of 1.0 +/- 0.1 ml/min, whereas liver had a clearance of 5.9 +/- 0.2 ml/min. Corresponding clearances in organs from 3 MC-pretreated rats were 8.9 +/- 0.5 and 6.7 +/- 0.6 ml/min for lung and liver, respectively. Small discrepancies between predicted and observed values could not be explained by non-uniform distribution of B[a]P or shunting of flow. These results suggest that enzyme kinetic data can be used to assess accurately the ability of lung and liver to clear xenobiotic compounds such as B[a]P and that, despite the great disparity in their metabolic capacity, under certain conditions these two organs may function equally well in the removal of circulating compounds from the blood.

Animals↗