Effect of wheat germ agglutinin and concanavalin A on insulin binding and response by Madin-Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, specific plasma membrane binding of [125I]insulin was undetectable. Correspondingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([ 3H]AIB) could be demonstrated. These results suggested that MDCK cells lack specific cell surface insulin receptors. To further examine this question intact MDCK cells were preincubated with antireceptor serum and subsequently labeled with [125I]protein A; however, insulin receptors were not detected. Control H4 hepatoma cells bound insulin, responded with increased glycogen synthesis and amino acid uptake, and possessed immunologically recognizable insulin receptor components. The insulin-associated response of stimulated [3H]AIB uptake was induced in MDCK cells by the insulinomimetic lectins concanavalin A (130-140% of basal value at concentrations of 10-40 micrograms/ml) and wheat germ agglutinin (140-160% of basal value at concentrations of 10-30 micrograms/ml). This stimulation was abolished by the respective lectin-specific monosaccharides D-mannose and N-acetyl-D-glucosamine. Together, these data indicate that the insulin-like activity of concanavalin A and wheat germ agglutinin can be elicited in MDCK cells even in the apparent absence of specific plasma membrane insulin-binding sites.

Transplantation of skin grafts in rats fed plant protein wheat gluten.

The effects of diets containing low biologic value plant protein wheat gluten and the high biologic value protein casein on skin allograft survival were studied in growing Charles River (C.D. strain) rats. Five groups of weanling rats were fed 16% casein or gluten with or without supplementation with the amino acids L-lysine and L-threonine. Allografts were obtained from the hooded Lister strain. Autograft experiments were also performed. The skin bed and the allografts were prepared by round-punch biopsies, and the grafts were retained in situ by cellophane tape dressings. In the gluten-fed rats, no rejection of allografts was observed during the 45-day study period. In the casein-fed rats, a 90% rejection rate occurred on Days 10-14 after transplantation. The absence of rejection in gluten-fed rats may be explained by an impaired cellular immune response due to protein malnutrition. Supplementing the gluten diet with the essential amino acids L-lysine and L-threonine restored the rejection rate to that of the control rats. The cell-mediated immune response, as measured by in vitro stimulation of lymphocytes with the mitogen concanavalin A, was severely impaired by the gluten diet. These experiments indicate that low biologic value protein diet in the rat is associated with skin allograft acceptance, probably due to impairment of cell-mediated immunity.

Humoral and cellular immune response in growing rats fed a 10% gluten diet.

The effect of protein malnutrition, induced by a diet containing 10% plant wheat gluten, on the immune response system was studied in growing rats. The rats were fed wheat gluten or casein (control) diets, and were the offspring of mothers fed a commercial stock rat diet, a gluten diet, or a casein diet during pregnancy and lactation. Rats fed dietary wheat gluten showed a depressed humoral immune response to antigenic stimulation with sheep red blood cells, regardless of the mother's diet. Both IgG and IgM antibody titers were depressed by the gluten diet, but the effect on IgG was more pronounced. Total hemolysin titers of gluten-fed rats were 41% that of the casein-fed rats. Hemolysin antibodies resistant to 2-mercaptoethanol appeared in the gluten-fed rats 5 days later than in the control rats, and their titers were 38% that of the controls. The number of spleen plaque-forming cells in the experimental rats at the peak of response was lower and delayed by 1 day. An impairment in the cellular immune response, measured by in vitro stimulation of lymphocytes with concanavalin A, was found in the gluten-fed rats. The amount of [3H]methylthymidine incorporated into newly synthesized DNA of lymphocytes taken from lymph nodes of rats fed gluten was 11% that of the control rats. These findings may suggest that a gluten diet, which induces protein malnutrition, affects thymus-dependent immune responses.

Insulin receptor regulation in minimal deviation hepatoma cell line.

Treatment of H4 hepatoma cells with the lectin wheat germ agglutinin (WGA) in the concentration range of 10-25 micrograms/ml increased 125I-insulin binding fivefold as compared to control binding in untreated cells. The increased insulin binding was rapid, readily reversible, and correlated with a 10-fold increase in the binding affinity of the receptor for insulin. Kinetic studies indicate that this increased affinity resulted from a decrease in the dissociation rate. The effect was specifically mediated by the lectin since it was reversed by simultaneous incubation with the monosaccharide N-acetyl-D-glucosamine (50 mM) or the disaccharide N,N'-diacetylchitobiose (1 mM). The WGA-mediated increase in insulin binding was not caused by inhibited insulin degradation. While WGA (5 micrograms/ml) mimicked insulin to induce stimulated uptake of [3H]aminoisobutyrate, the lectin failed to enhance the biological sensitivity of H4 hepatoma cells to insulin. At higher concentrations of WGA (125 micrograms/ml), interference with the insulin-mediated response was observed. Trypsin treatment of H4 hepatoma cells prior to measuring binding of 125I-insulin in the presence of increasing concentrations of native insulin, led to a leftward shift of the competition curve, indicating an increased affinity of the receptor. No further increase was observed when the trypsin-treated cells were subsequently exposed to WGA. These results suggest that trypsin treatment and WGA exposure may increase the affinity of the receptor by a similar mechanism. The results are consistent with the concept that WGA and trypsin decrease interaction between insulin binding and receptor affinity regulating components in the plasma membrane, leading to an increase in the affinity of the receptor for insulin.

Insulin-ricin B hybrid molecules: receptor binding and biological activity in a minimal-deviation hepatoma cell line.

Hybrid molecules were produced by covalently coupling the hormone insulin to the binding chain B of the plant toxin ricin. Binding of the insulin-ricin B hybrid to minimal-deviation hepatoma cells occurred primarily through ricin-specified cell-surface carbohydrates (galactose, N-acetylgalactosamine) since 125I-insulin-ricin B binding to cells could be 90% displaced by 50 mM lactose. [14C]Glucose incorporation into glycogen was maximally stimulated approximately 80% by insulin, whereas maximum stimulation by insulin-ricin B hybrid was greater than 100%. Ricin B chain alone was non-stimulating at concentrations tested (10(-9)-10(-7) M). Furthermore, the stimulation of [14C]glycogen labeling mediated by the hybrid was markedly inhibited by 1 mM lactose, while this sugar had no effect on the stimulation mediated by native insulin. Additionally, a preparation of ricin B shown to actively displace up to 80% of the binding of 125I-hybrid to cells also inhibited hybrid-mediated [14C]glycogen production. These results indicate that insulin-ricin B hybrid molecules possess toxin-specified binding abilities while evoking the insulin-associated cellular response of stimulated incorporation of [14C]glucose into glycogen. Such results thus suggest the possibility that alternate cell-surface receptors may play a role in conveying insulin's intracellular metabolic-control signals.

Insulin-ricin B hybrid molecules mediate an insulin-associated effect on cells which do not bind insulin.

A hybrid molecule was constructed by covalently linking, by a disulfide bridge, the hormone insulin to the binding subunit B of the plant toxin ricin (specificity: Gal, GalNAc). Monolayer-cultured MDCK cells, which lack detectable levels of specific plasma membrane 125I-insulin binding but which readily bind 125I-insulin-ricin B, were used in these studies. Binding of insulin-ricin B to these cells could be displaced by lactose and ricin B, but not by insulin. The biological activity of the hybrid, as measured by [14C]glucose incorporation into glycogen, was stimulated in a dose-dependent manner by the hybrid (10(-11)-10(-8) M), whereas glycogen production was not stimulated by insulin alone. The stimulated glycogen labeling in response to the hybrid was also inhibited by lactose and ricin B. When ricin B alone was tested over the same range of concentrations, stimulation of glycogen synthesis was not observed, nor was there any evidence for stimulation when insulin and ricin B were added simultaneously. These data suggested that alternate cell surface receptors (i.e. ricin B receptors) may substitute for specific receptors (i.e. insulin receptors) to convey intracellular metabolic control signals in this cell line.