Guinea Pig Anti-insulin Serum Research Articles

Insulin dependence of murine lymphoid T‐cell leukemia

The in vitro proliferation of the spontaneous lymphoid T-cell leukemia designated LB was enhanced by physiological, intermediate and supraphysiological concentrations of insulin. The enhancing effect was observed in both serum-free medium (SFM) and medium containing low concentrations of serum. Guinea-pig anti-insulin serum, but not guinea-pig normal serum, inhibited the proliferation of LB cells incubated either in medium containing serum alone or in medium containing serum and supplemented with insulin. This finding suggests that LB cells use serum insulin as a growth factor. Insulin-like growth factors I (IGF-I) and II (IGF-II) failed to stimulate an appreciable proliferation in LB cells, whereas in the same experiment insulin markedly enhanced the proliferation of this lymphoid leukemia. Furthermore, the concentration of unlabelled insulin required to displace 50% of 125I-insulin bound to LB cells was 3 orders of magnitude lower than the concentration of IGF-I required to achieve the same displacement. Our findings indicate that interaction of insulin with its own receptor, and not with IGF-I receptor, triggers the proliferation of LB cells. Radio-receptor assays revealed that LB cells express approximately 3,200 molecules of high affinity (Kd = 10(-9) M) insulin receptor per cell. None of 7 other tumor cell lines tested responded to insulin. The proliferation of insulin-stimulated LB cells was also inhibited with tyrphostin, a tyrosine kinase blocker analogous to tyrosine, which perhaps blocks the tyrosine kinase activity of the insulin receptor beta-chain.

A novel enzyme immunoassay of anti-insulin IgG in guinea pig serum

A novel enzyme immunoassay of anti-insulin IgG in guinea pig serum is described. Guinea pig anti-insulin serum diluted with nonspecific guinea pig serum was incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate and a rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. After washing to eliminate nonspecific guinea pig IgG in the diluted serum, the polystyrene ball was incubated with dinitrophenyl-L-lysine to elute the complex of anti-insulin IgG and the conjugate. The eluate was incubated with an avidin-coated polystyrene ball. Finally, the amount of guinea pig anti-insulin IgG in the complex trapped onto the avidin-coated polystyrene ball was measured by incubation with rabbit (anti-guinea pig IgG) Fab′-peroxidase conjugate. This enzyme immunoassay was 10,000-fold more sensitive than the conventional enzyme immunoassay using insulin-coated polystyrene ball and rabbit (anti-guinea pig IgG) Fab′-peroxidase conjugate.

Scintigraphic distribution of complexes of antiinsulin antibodies and 123I-insulin. In vivo studies in rats.

Clearance of immune complexes made of antiinsulin antibodies and 123I-insulin was studied with scintillation scanning in anesthetized rats. Complexes made with purified guinea pig antiinsulin IgG2 (cytophilic isotype) were rapidly cleared by the liver whereas those made with IgG1 remained in the plasma, as did 123I-labeled IgG1 or IgG2 of control animals. Hepatic clearance of insulin-antiinsulin IgG complexes was not inhibited by either an excess of insulin or decomplementation, thereby ruling out interaction with insulin and C3b receptors. Insulin and guinea pig antiinsulin serum or its purified IgG isotypes formed large aggregates exceeding 5 IgG. Antiinsulin antibodies of diabetics, mostly IgG1 and IgG3 (cytophilic isotypes), formed complexes that either remained in plasma (small aggregates) or were cleared by the liver (large aggregates). In conclusion, clearance of insulin-antiinsulin IgG complexes is probably mediated by Fc gamma receptors on macrophages and requires cytophilic subclass composition and formation of large IgG aggregates.

Acute effects of starvation and treatment of rats with anti-insulin serum, glucagon and catecholamines on the state of phosphorylation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in vivo.

The fraction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the dephosphorylated (active) form in rat liver in vivo was measured after various experimental treatments of animals. Intraperitoneal injection of glucose (to raise serum insulin concentrations) into rats 4 h into the light phase (L-4) resulted in a transient (30 min) increase in the expressed (E)/total (T) activity ratio of HMG-CoA reductase without any change in total activity (obtained after complete dephosphorylation of the enzyme). Conversely, intravenous injection of guinea-pig anti-insulin serum into rats 4 h into the dark phase (D-4) significantly depressed the E/T ratio within 20 min. Intravenous injection of glucagon into normal rats at this time point did not affect the degree of phosphorylation of the enzyme, in spite of a 10-fold increase in hepatic cyclic AMP concentration induced by the hormone treatment. A 3-fold increase in the concentration of the cyclic nucleotide induced by adrenaline infusion was similarly ineffective in inducing any change in expressed or total activities of hepatic HMG-CoA reductase. However, when insulin secretion was inhibited, either by the induction of streptozotocin-diabetes or by simultaneous infusion of somatostatin, glucagon treatment was able to depress the expressed activity of HMG-CoA reductase (i.e. it increased the phosphorylation of the enzyme). Therefore insulin appears to have a dominant role in the regulation of the phosphorylation state of hepatic HMG-CoA reductase. In apparent corroboration of this suggestion, short-term 4 h food deprivation of animals before D-4 resulted in a marked decrease in the E/T activity ratio of reductase, which was not affected further by an additional 8 h starvation. By contrast, the total activity of the enzyme was not significantly affected by 4 h starvation, but was markedly diminished after 12 or 24 h starvation. Longer-term starvation also produced a chronic increase in the degree of phosphorylation of the enzyme. These results are discussed in relation to the role of reversible phosphorylation in the control of hepatic HMG-CoA reductase activity in vivo.

Insulin regulation of rat growth hormone gene transcription.

We have previously shown that insulin suppresses growth hormone (GH) messenger (m) RNA levels in rat pituitary cells. To further delineate the molecular mechanism of insulin action, the effect of insulin treatment on GH gene transcription rates was examined in GH3 pituitary cells grown in serum-free defined medium. A transcriptional run-off assay was performed when intact isolated nuclei were allowed to continue RNA synthesis in an in vitro reaction. Specific incorporation of [32P]GTP into RNA was quantified by hybridization to rat GH complementary (c) DNA. Hybridization efficiency was measured with an internal [3H]cRNA standard and ranged from 30 to 48%. Alpha-amanitin (1 microgram/ml) inhibited total transcription, and excess unlabeled rat pituitary mRNA (250 ng) competitively inhibited GH mRNA hybridization by greater than 80%. Insulin (0.7 nM) inhibited new GH mRNA synthesis, and maximal inhibition (30% of control) was observed with 7 nM insulin after 4 h treatment. The inhibitory effects of insulin on new GH mRNA synthesis were abolished by both insulin-receptor-antiserum and by guinea-pig anti-insulin serum. The results show that insulin exerts a rapid suppression of new GH mRNA synthesis. These data provide evidence for the direct transcriptional regulation of the GH gene by insulin.

A Highly Sensitive Enzyme Immunoassay of Anti-Insulin Antibodies in Guinea Pig Serum

Abstract A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. Insulin bound to anti-insulin antibodies was separated from free insulin by precipitation with polyethylene glycol. Anti-insulin antibodies in the precipitates were dissociated from insulin and inactivated by incubation with 0.1 mol/1 HCl. The amount of insulin dissociated was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab′-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was improved 1,000-fold as compared with that of the enzyme immunoassay previously described, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab′-horseradish peroxidase conjugate.

Insulin within islets is a physiologic glucagon release inhibitor.

To determine if glucagon secretion is under physiological control of intra-islet insulin, pancreata from normal rats were perfused at a 100 mg/dl glucose concentration with either guinea pig antiinsulin serum or normal guinea pig serum in a nonrecirculating system. Perfusion of antiserum was followed within 3 min by a significant rise in glucagon that reached peak levels three times the base-line values and assumed a hectic pattern that returned rapidly to base-line levels upon termination of the antiserum perfusion. Nonimmune guinea pig serum had no effect. To gain insight into the probable site of insulin neutralization, 125I-labeled human gamma-globulin was added to antiserum or nonimmune serum and perfused for 3 min. More than 83% of the radioactivity was recovered in the effluent within 3 min after termination of the infusion, and only 0.05 +/- 0.015% of the radioactivity injected was present in the pancreas 10 min after the perfusion. The maximal amount of insulin that could be completely bound to insulin antibody at a dilution and under conditions simulating those of the perfusion experiments was 20 mU/min. It is concluded that insulin maintains an ongoing restraint upon alpha cell secretion and in its absence causes hectic hypersecretion of glucagon. This restraint probably occurs largely in the intravascular compartment. Loss of this release-inhibiting action of insulin may account for initiation of hyperglucagonemia in insulin-deficient states.

Insulin suppresses growth hormone secretion by rat pituitary cells.

The effects of insulin on basal and hydrocortisone-induced growth hormone (GH) secretion were studied in rat pituitary tumor cells (GH3). Cells were grown in monolayer culture and exposed to exogenously added insulin for up to 8 d. Basal GH secretion was inhibited by insulin (0.7 nM) after a 48-h lag period by approximately 50% (P less than 0.01, vs. untreated control cells). The suppression of GH secretion was reversible, as removal of added insulin resulted in return of GH secretion to normal levels after 24 h. Maximal suppression of basal GH secretion was achieved by 0.7 nM insulin, and these effects were prevented by simultaneous exposure of the cells to guinea pig anti-insulin serum (1:2,000). No effects of insulin on cell replication were evident, and glucose concentration in the medium did not differ in control or insulin-treated wells. Insulin (7 nM) significantly suppressed the fivefold hydrocortisone-induced GH stimulation during 5 d of incubation with up to 1,000 nM of the steroid (P less than 0.001). These inhibitory effects were similarly observed in glucose- and pyruvate-free medium, and in the presence of 2-deoxyglucose. Insulin also reversed the suppression of prolactin (PRL) secretion induced by hydrocortisone (1 uM), and actually stimulated basal PRL secretion by over 50%. Insulin did not alter the inhibitory effect of hydrocortisone on GH3 cell proliferation. Although higher doses (13 nM) of insulin-like growth factor (IGF-I) also suppressed basal GH secretion, IGF-I did not alter the GH and PRL secretory changes induced by hydrocortisone. The results show that insulin exerts a direct, specific inhibitory effect on basal and hydrocortisone-induced GH secretion by GH3 cells unrelated to glucose utilization by the cells.

Effects of glucose, insulin and nicotinic acid on adipose tissue blood flow in rats.

Adipose tissue blood flow (ATBF) was examined in rat parametrial fat by the 133-Xe elimination method. Intravenous infusion of glucose to fed rats resulting in blood glucose concentrations of 10-12 mmol X 1(-1) caused a significant reduction in ATBF (-37%). Similar infusions to 48 hour fasted rats had no consistant effect on ATBF. Glucose infusion caused a significant rise in plasma insulin concentrations in both fed and fasted animals, although the average concentration in fasted rats given glucose did not exceed the control value in fed animals. Insulin added to the glucose infusion caused a similar reduction in ATBF in fasted animals as that seen after glucose alone in fed animals (-38%). Guinea pig anti insulin serum administered intravenously to fed rats elicited an increase in blood glucose concentrations similar to that seen after glucose infusion, but was without effect on ATBF. These results suggest that the effect of glucose on ATBF is secondary to a release of insulin resulting in plasma levels above those found in fed control rats. Infusion of nicotinic acid also reduced ATBF without influencing blood glucose concentration and in spite of insulin concentrations lower than in fed control rats. Since both insulin and nicotinic acid inhibit the formation of c-AMP in adipocytes, it is hypothesized that both compounds decrease ATBF by decreasing the release of the vasodilator adenosine from the cells.

Biological activity of rat proinsulin synthesized by Escherichia coli

Fused proteins possessing insulin antigenic determinants are synthesized and secreted by several strains of Escherichia coli K-12 bearing plasmids containing cDNA copies of rat proinsulin mRNA. Small amounts of the secreted, fused protein from E. coli strains χ1776 and HB101 bearing plasmid p 147 were partially purified and assayed for biological activity by determining stimulation of [1- 14C]glucose oxidation to 14CO 2 in the rat epididymal fat pad assay. Guinea pig anti-insulin serum (GPAIS) was used to suppress glucose oxidation stimulated by insulin. After mild treatment with trypsin, fused protein stimulated 14CO 2 production, and over 90% of this activity was suppressible by GPAIS. Untrypsinized fused protein demonstrated no GPAIS-suppressible 14CO 2 production. Larger amounts of fused protein were obtained by transformation of strain PR 13 of E. coli in which the yield of eukaryotic proteins is increased approx. 50-fold. A single intravenous injection of trypsinized fused protein from E. coli PR13 containing plasmid p287.47 resulted in a rapid decline in the plasma glucose levels of fasted mice, and the hypoglycemic effect persisted for at least 120 min.

Pentosephosphate Shunt Activity of Rat Pancreatic Islets: Its Dependence on Glucose Concentration*

Although several attempts have been made to establish a relationship between glucose concentration and pentosephosphate shunt (PPS) activity of pancreatic islets in vitro, no such relationship has been found. Recently it has been shown that exogenous insulin, when added in vitro, inhibited islet PPS activity. Since it was possible that the failure to detect such a relationship was due to insulin released into the incubation medium, we reinvestigated the subject, introducing GPAIS (guinea pig antiinsulin serum) to bind insulin in the medium. When five islets were incubated with 16.7 mM glucose for 90 min, the absolute rate as well as the percentage of islet PPS activity were found to be significantly higher (P less than 0.05) in the presence of GPAIS than in its absence. In the presence of GPAIS, the absolute rate and the percentage of islet PPS activity increased in a dose-related manner (18, 43, and 124 pmol/five islets.90 min and 2.3%, 3.2%, 9.4%, respectively) when the glucose concentration was raised from 5.6 to 11.1 and 16.7 mM. Our data, therefore, indicate that the activity of islet PPS activity in vitro depends on the concentration of glucose, provided that insulin does not accumulate in the incubation medium. This suggests that PPS activity might play a role in glucose-induced insulin secretion. However, whether the PPS contributes to the secretory mechanism by producing a signal for insulin secretion or by forming cofactors required by the secretory mechanism remains to be established.

The metabolism of oleic acid by the perfused rat liver in experimental diabetes induced by antiinsulin serum

The metabolism of varying quantities of oleic acid was examined in isolated perfused livers from normal fed rats and from animals made diabetic by pretreatment with guinea pig antiinsulin serum (AIS). The data presented reemphasize the fact that the quantity of free fatty acid (FFA) coming to the liver is a necessary, but not the most important, factor affecting the subsequent metabolism of the FFA. Rates of ketogenesis and output of triglyceride and the terminal concentration of hepatic triglyceride were proportional to uptake of FFA in certain concentration ranges. For equal rates of uptake of FFA, ketogenesis was greater, and the quantity of triglyceride secreted or accumulated within the liver was less, with livers from diabetic animals than with livers from normal animals. In confirmation of previous data, the liver was observed to have a maximal capacity to secrete triglyceride. Triglyceride accumulated in livers from normal-fed and diabetic animals only when uptake of FFA was more than sufficient to saturate the secretory process. Since proportionately more FFA was catabolized by livers from AIS treated animals, greater uptake of FFA was required to produce maximal rates of output of triglyceride and accumulation in livers from diabetic than from normal animals. Rates of ketogenesis by livers from normal fed animals increased minimally with increasing uptake of FFA (up to 1.0 m M free fatty acid). Even when uptake increased considerably with FFA concentrations of approximately 2.5 m M, rates of ketogenesis by livers from normal animals were less than half those of livers from diabetic rats, and maximal rates were not achieved by the normal controls. It is evident that changes in hepatic metabolism of FFA in the intact diabetic animal result from simultaneous alterations of supply of FFA and hormonally induced metabolic changes in the liver. Moreover, although hepatic secretion and accumulation of triglyceride is greater in isolated perfused livers from normal rats than from diabetic animals when the livers are exposed to equal quantities of FFA, the diabetic livers can accumulate more triglyceride, secrete more triglyceride, and oxidize more FFA to ketone bodies than can the normal under conditions in which considerably more substrate is available to the diabetic rather than to the normal livers. These differences might also be expected to occur in the acutely insulin deficient intact animal, in which changes in hormonal status and substrate (FFA) availability occur simultaneously, and might, in part, explain the ketonemia, hypertriglyceridemia, and hepatic steatosis often observed in vivo.

Effects of anti-insulin serum, insulin, and glucose on output of triglycerides and on ketogenesis by the perfused rat liver.

The rate of change of the concentration of various metabolites in blood in vivo and of the metbolism of free fatty acids by the perfused liver in vitro was sutidied as a function of time after the induction of acute insulin deficiency in rats by administration of guinea pig anti-insulin serum; the rate of reversal of these changes afte treatment of the anti-insulinserum diabetic ratss with insulin was also investigated. The concentrations of blood glucose and ketonebodies, and plasma-free fatty acids increased rapidly after injection of anti-insuli serum, while plasma triglycerides increased more slowly. These alterations were restored rapidly toward normal after treatment of the diabetic animals with insulin...

Insulin binding to plasma membranes

Insulin binding to plasma membranes from rat epididymal fat pad, liver, brain and human red blood cell membranes was estimated by the displacement of [ 125I]-insulin by native insulin. Membranes isolated from rat epididymal fat pad bound more insulin than rat liver plasma membranes. Rat brain and human red cell membranes bound essentially no insulin. Glucose bound to fat pad membranes and this binding was decreased by insulin. The binding of labeled insulin was reduced in proportion to the relative concentration of labeled and unlabeled insulin. Maximum insulin binding required the presence of Na +, K + and glucose. The binding of insulin was not altered by the insulin derivatives: sulfonated B chain, reduced-reoxidized insulin and oxidized B chain. The binding of insulin was inhibited in proportion to the concentration of added guinea pig anti-insulin serum. An increase in insulin binding was obtained following treatment of fat pad membranes with either phospholipase C or trypsin.

Early effects of anti-insulin serum on hepatic metabolism of plasma free fatty acids in dogs.

SUMMARY:Hepatic metabolism of free fatty acids was studied in six anesthetized fasted dogs before and for several hours after the abrupt induction of insulin deficiency. This was produced by injecting guinea pig anti-insulin serum in amounts capable of binding all circulating insulin for several hours. During each study, albumin-bound-I-C-14-palmitate and I-131-rose bengal were infused at a constant rate and serial sanv pies of arterial, portal and hepatic venous blood were obtained through appropriate intravascular catheters. Maximal changes were observed three to four hours after administration of anti-insulin serum. Mean arterial concentrations of glucose, free fatty acids, glycerol and triglyceride fatty acids of very low density lipoproteins increased twofold and that of ketone bodies, fourfold. Hepatic extraction ratio of free fatty acids was unchanged after anti-insulin serum so that the hepatic uptake of free fatty acids approximately doubled. During the control period, about 25 per cent of free fatty acids removed by the liver were converted to CO2, 11 per cent to triglyceride fatty acids of very low density lipoproteins and 25 per cent to ketones (assuming that ketones derived entirely from FFA). After anti-insulin serum, corresponding values were 14 per cent, 26 per cent and 51 per cent. The increased relative and absolute conversion of free fatty acids to ketones resembles that observed in chronically diabetic dogs withdrawn from insulin for forty-eight hours. However, the increased conversion of free fatty acids to triglyceride fatty acids of very low density lipoproteins observed in short-term deficiency of insulin contrasts with the greatly depressed conversion observed after two days of insulin lack.

Effect of phentolamine upon insulin secretion during exercise.

Rats were injected with guinea-pig antiinsulin serum and131I-labelled human albumin. The amount of endogenously secreted insulin was estimated from the progressive reduction in the pool of circulating unneutralized antibodies. In animals compelled to swim, insulin secretion occured at a much lower rate than in control rats. Prior injection of phentolamine abolished the exercise-induced inhibition of insulin release. These findings support the concept that such inhibition is dure to endogenously released catecholamines.

Effects of insulin and anti-insulin serum treatments on levels of metabolites in rat mammary glands.

The metabolic lesions produced by insulin insufficiency in lactating Sprague-Dawley rats were characterized. Guinea pig anti-insulin serum was used to produce acute insulin insufficiencies in lactating rats. Metabolite level changes in mammary tissue of animals receiving anti-insulin serum were observed. These changes were discussed in relation to activities of the Embden-Meyerhoff and pentose phosphate pathways. Calculations of the redox state of free nicotinamide adenine dinucleotides indicated that insulin insufficiency produced a shift in the redox state in mammary tissue. In addition, acute diabetes resulted in a decrease in the proportion of high energy phosphate bonds to the total adenine nucleotides in mammary tissue. These observations suggest that insulin's influence on mammary tissue metabolism was manifested primarily through its effects upon the redox state of free nicotinamide adenine nucleotides. This effect could possibly be attributed to reduced activity of the electron transport system. ...

Circulating insulin: th proinsulin-like properties of "big" insulin in patients withou islet cell tumors.

When plasma is filtered on Sephadex G-50, insulin immunoreactivity is recovered in two peaks. "Big" insulin, the higher molecular weight component, and "little" insulin, the lower molecular weight component, have elution volumes that correspond to those of proinsulin-(125)I and insulin-(125)I respectively. When plasma was extracted with acid ethanol and filtered in 1.0 M acetic acid, the patterns and proportions of "big" and "little" insulin were indistinguishable from those obtained by filtration of whole plasma in neutral buffer. When "big" insulin was isolated from plasma and mixed with a tracer of porcine proinsulin-(125)I, trypsin converted the "big" insulin immunoreactivity to the gel filtration pattern of "little" insulin in the same way that it converted the proinsulin radioactivity. More than 90% of both "big" insulin and proinsulin were converted at optimal trypsin concentrations. Our present guinea pig anti-insulin serum failed to distinguish "big" from "little" but a porcine proinsulin anti-serum, under appropriate conditions of assay, reacted strongly with "big" insulin but not at all with "little." When tested on isolated fat cells, "little" insulin had the same bioactivity as porcine insulin, whereas "big" insulin had the same low activity as porcine proinsulin. These studies suggest that "big" insulin represents either single-chain proinsulin and/or a proinsulin intermediate that has similar low bioactivity.

Reactions of proinsulin and its derivatives with antibodies to insulin.

SummaryThe reactions of porcine proinsulin and its degradation products with insulin antibodies in guinea pig anti-insulin serum can be shown to vary with the immunological system used for study. From the results obtained with two such systems it is concluded that the connecting chain of amino acids in the molecule of proinsulin must mask sites on the insulin molecule which are capable of reacting with insulin antibodies.

Delayed Hypersensitivity to Insulin and Its Component Polypeptide Chains

Abstract Bovine insulin produces a state of pure delayed hypersensitivity in guinea pigs from the 7th to the 13th days after sensitization with 10 µg of insulin in Freund's complete adjuvant. A delayed response in insulin-sensitized animals was readily elicited with insulin and with the isolated B-chain of insulin, while the A-chain, at equivalent doses, elicited only marginal reactions. Desensitization experiments wherein guinea pigs were immunized with insulin, B-chain or A-chain showed that each of these molecules contains determinants capable of inducing and eliciting a delayed response, and that at least one locus on the isolated B-chain produces delayed reactivity which can be elicited with the intact insulin molecule. Guinea pig anti-insulin serum was not reactive with isolated A- or B-chain. The use of insulin as a model protein for analysis of the relative contribution of stereochemical configuration in producing the immediate as opposed to the delayed immune response is discussed.