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Lactation in goats has been shown to modify in vivo insulin action. [Debras, E., J. Grizard, E. Aina, S. Tesseraud, C. Champredon, and M. Arnal. Am. J. Physiol. 256 (Endocrinol. Metab. 19): E295-E302, 1989]. To further elucidate the mechanism of insulin action, we studied insulin binding and insulin receptor tyrosine kinase activity in solubilized and partially purified receptor preparations from liver and skeletal muscles (longissimus dorsi, tensor fascia lata, diaphragm, and masseter) from lactating and nonlactating goats. Lactation did not alter insulin receptors in the various skeletal muscles and had a minor influence on liver receptors (where only a 20% increase in receptor number was visible, P less than 0.05). Insulin-stimulated autophosphorylation and the kinase activity against polyglutamyltyrosine (4:1) were not significantly modified in skeletal muscle receptor preparations from lactating goats when compared with nonlactating animals. They tended to decrease in liver preparations, but not significantly. Thus the changes in insulin action in vivo during lactation in goats were not related to modifications in insulin kinase activity but were probably localized at a postreceptor level.

The influence of protein content of the diet on the plasma concentrations and binding to skeletal muscle and liver of insulin and insulin-like growth factor-1 (IGF-1), was studied in growing rats. Animals with a starting body-weight of 80 g received for an 11 d period isoenergetic diets containing (g/kg dry matter) 155 protein as controls (MP), or 55 (LP) or 300 (HP) protein. Food was offered as six equal meals/d. Daily food intakes provided adequate amounts of energy. Total plasma IGF-1 increased linearly as a function of dietary protein intake. Plasma insulin was lower in the LP than in the MP and HP groups. Hormone binding was studied in wheat-germ agglutinin (WGA) partially purified skeletal muscle receptor preparations. Each 125I-labelled hormone binding was competed for by increasing amounts of homologous and heterologous unlabelled hormone; this displacement needed lower concentrations of homologous than heterologous hormone. When compared with MP-diet feeding, the LP diet resulted in an increased ligand concentration for half-maximal binding. In addition the specific 125I-labelled insulin and 125I-labelled IGF-1 binding increased at all hormone concentrations and, as revealed by Scatchard analysis, the hormone binding capacity also rose (only significant for low-affinity insulin receptors and high-affinity IGF-1 receptors). The HP diet had little effect on hormone binding, except to increase insulin binding at very low insulin concentrations. Hormone binding was further studied in WGA partially purified liver receptor preparations. Those preparations did not exhibit any detectable specific 125I-labelled IGF-1 binding. The specific 125I-labelled insulin binding was not altered by dietary protein level. It is concluded that the increase in skeletal muscle insulin and IGF-1 binding along with a decrease in insulin and IGF-1 in the blood from rats fed on the LP diet, is consistent with the concept of an inverse relationship between plasma hormone and hormone binding. The physiological significance with respect to metabolic adaptation of muscle remains to be established.

The effect of specific calorie deprivation was studied in meal-fed growing rats. It resulted in a 50% decrease in growth rate. Blood glucose and most non-essential blood free amino acid levels were depressed. Postprandial plasma insulin was decreased. With insulin ranging from 0.01 to 100 nM, insulin binding to crude Triton X-100 solubilized membranes from liver was higher in calorie restricted rats when compared with control rats. Wheat germ agglutinin (WGA) purified receptor preparations also exhibited higher insulin binding in liver from experimental group but the significance (P less than 0.05) was only visible with low insulin levels; both basal and insulin-stimulated tyrosine-kinase activity were left unchanged. In contrast, whatever the skeletal muscle insulin receptor preparation (enriched plasma membranes, crude Triton X-100 solubilized or wheat-germ agglutinin purified extracts) insulin binding was similar in control and calorie-restricted rats.

Insulin binding and insulin receptor kinase activity were measured in solubilized and partially purified receptor preparations from liver and skeletal muscles of rats that were either fed a standard diet or subjected to a 72-hour fasting period. Insulin binding capacity was increased in both tissues from fasted rats as determined by Scatchard analysis. The affinity of the receptors was not modified by fasting. Affinity labeling of the alpha-subunit of insulin receptors also suggested an increase in the number of insulin receptors in both tissues. The ability of insulin to stimulate the autophosphorylation of the beta-subunit as well as the phosphorylation of the artificial substrate Glu80-Tyr20 was significantly impaired in liver from fasted rats and by contrast unchanged in skeletal muscles. These findings indicate that in rats, fasting produces changes in insulin receptor kinase activity in liver but not in muscle. The physiological significance of this tissue-specific regulation of receptor kinase activity in relation to insulin action during fasting remains to be established.

Insulin/glucagon control of hepatic metabolism, i.e. a endocrine-nervous system, is one of the general systems of integration in vertebrates. In this system, substrates coming from the digestive tract or from extrahepatic metabolism are important messenger molecules. Liver uptake of insulin and glucagon mainly accounts for high metabolic clearance rates of these hormones in both ruminants and non-ruminants. Glucagon infusion into ruminants results in an increase in the net hepatic uptake of glucose precursors and gluconeogenesis. Glucagon effects have also been demonstrated in isolated hepatocytes. Glucagon, through its effect on pyruvate carboxylase (EC 6.4.1.1.) may regulate gluconeogenesis. Insulin infusion induces hypoglycaemia. As a result, glucagon secretion increases and counterregulates insulin action. However, it has been shown that hepatic gluconeogenesis decreases during euglycaemic hyperinsulin clamp, mainly due to a decrease in the hepatic supply of glucose precursors following insulin action in extrahepatic tissues. Insulin fails to elicit any significant effect in vitro. Hepatocytes exhibit insulin and glucagon receptors. The apparent characteristics of hormone binding in vitro are similar in ruminants and non-ruminants, but the characteristics of postreceptor events are unknown in the former. Glucagon, which influences hepatic glucose synthesis, may be a major hormone in ruminants.

Insulin-related material was measured in acid ethanol extracts of brain, testis, liver, and kidney from adult rats acutely injected with insulin or saline. Insulin injection resulted in a twofold to threefold increase in plasma insulin during a two-hour period after injection. Plasma glucose was greatly depressed. Insulin injection had no effect on the insulin-related material in most areas of brain (cerebral cortex, olfactory bulbs, and medial hypothalamus) and the cerebrospinal fluid; lateral hypothalamus was an exception and paradoxically exhibited a decrease of this material. The testis insulin-related material was unaffected; purification of the testis extracts using the C18 Sep pak method revealed no further difference between the animals. In liver, the insulin-related material was not significantly different in the control and the insulin-injected group; however, we found a significant correlation between this material and plasma insulin within the insulin-injected group. In contrast, insulin injection resulted in an important increase in kidney insulin-related material that paralleled the change in plasma insulin. Thus, like chronic experiments, acute hyperinsulinemia revealed that the insulin-related material was largely independent from blood insulin in tissues that exhibit very different insulin uptake from the blood; kidney appeared to be an exception.

The fate of plasma glucagon has been analyzed in detail by Desbuquois and Postel-Vinay. The present work was carried out to clarify the relationships between plasma glucagon disappearance and its tissue uptake. For the purpose, we injected rats intravenously with 125I-glucagon alone or with concomitant or sequential injections of native glucagon. Plasma 125I-glucagon was analyzed by Biogel P10 chromatography. Liver and kidney glucagon kinetics were studied from the point of view of the evolution of the total radioactivity present in each tissue a few minutes after glucagon injection. 125I-glucagon was rapidly cleared from the plasma (half-life within 2 min); it was intensively associated with liver and kidneys. Liver radioactivity rapidly declined within the first 5 min after injection, whereas kidney radioactivity increased. The concomitant injection of increasing amounts of native glucagon with 125I-glucagon progressively reduced the liver radioactivity, indicating that glucagon was trapped in a saturable compartment. In contrast, kidney radioactivity remained unchanged. The sequential injection of 125I-glucagon and excess native glucagon resulted in a shift to the right in the plasma 125I-glucagon decay curve which suggests that the glucagon excess displaced 125I-glucagon from its distribution compartment back into the plasma. The compartment where glucagon uptake occurred a few minutes after 125I-glucagon injection displayed some of the fundamental properties of glucagon receptors, i.e. saturatibility and reversibility.

The purpose of this work was to investigate liver glucagon receptors in growing rats fed a control diet (11.8% crude protein) or a high-protein diet (19.8% crude protein) given in restricted amounts. The animals were fed every 4 hours. 125I-glucagon binding to purified liver plasma membranes was studied. Membrane purity was analysed with marker enzymes. The alteration of glucagon during incubation was measured. The results show that specific 125I-glucagon binding increased with time at 30 degrees C, reaching a maximal value within 120 min. The increasing level of unlabelled glucagon inhibited 125I-glucagon binding at steady state. Apparent specific 125I-glucagon binding at steady state was lower in experimental animals than in controls. This correlated with the increase in glucagon breakdown and decrease in membrane purity. Alternatively, glucagon binding to its receptors could drop. Unlabelled glucagon excess produced a time-dependent dissociation of glucagon-receptor complexes (half-life: up to 1 h). Feeding the experimental diet increased the dissociation of labelled glucagon-receptor complexes.

Insulin binding and tyrosine kinase activity of the insulin receptor have been measured in the liver and muscles of rats fed or submitted to a 72-h-fasting. In both tissues, insulin binding increased in fasting rats. In liver, the ability of insulin to simulate receptor tyrosine kinase activity greatly unpaired during fasting, but remained unchanged in muscle. The change during fasting of the insulin-stimulated tyrosine kinase activity of the insulin receptor is specific to certain tissue.

125I-glucagon binding and degradation were studied in highly purified plasma membranes from rat livers. Specific 125I-glucagon binding increased rapidly with time at 30 degrees C and reached a maximum between 30 and 120 min. At 120 min the labelled material present in the supernatants from incubation mixtures had extensively lost its ability to rebind to fresh membranes whatever the glucagon concentration. This impairment was not due to the release of a degradative activity into the incubation mixture, suggesting a membrane-mediated process. The presence of proteinase inhibitors (bacitracin/aprotinin) resulted both in an increase in specific 125I-glucagon binding to membranes and an improvement in the ability of the labelled material from the supernatant to rebind to fresh membranes. When analysed by Bio-Gel P-10 chromatography the loss in the ability of the labelled material in the supernatants to rebind to fresh membranes correlated with a decrease in the labelled material which eluted as 125I-glucagon from the column. Chromatographic analysis overestimated 125I-glucagon when compared to the radioreceptor assay. The labelled material extracted from membranes by Triton X-100 solubilization or dissociated from membranes after exposure to an excess of unlabelled glucagon mainly eluted as 125I-glucagon. However, a significant amount (20-30%) of the labelled material eluted in the low molecular weight region.

Binding of glucagon and glucagon-stimulated cyclic AMP production were studied in highly purified liver plasma membranes from growing rats fed a 12% protein diet (group 1) or 20% protein diet; this latter was given either in normal (group 2) or restricted (group 3) amounts. Groups 2 and 3 exhibited significantly higher peripheral glucagonemia than group 1 (amounting to 286 and 160% of group 1, respectively). Specific [125I]iodoglucagon binding to plasma membranes was similar in all groups. Scatchard analysis revealed no further differences between affinity constants and binding capacities in the three groups. Hormone degradation was constant. As membrane recovery and membrane purity were unaltered, these results suggest that hyperglucagonemia caused by increasing dietary protein level is not associated with any significant modification of glucagon binding sites in rat liver. In the presence of a potent phosphodiesterase inhibitor (3-isobutyl-1-methyl xanthine 0.2 mM) the glucagon-stimulated cyclic AMP production was higher in rats fed the 20% protein diet, which was given in normal amounts, than in rats fed the 12% protein diet. In contrast when the 20% protein diet was given in restricted amounts the glucagon-stimulated cyclic AMP production was similar to that in the 12% protein-fed rats.