High-carbohydrate and high-protein diets alter liver and brain insulin receptors in rats

Insulin receptors of liver and brain plasma membranes were investigated in rats kept on standard (S), high-carbohydrate (HC) and high-protein (HP) diets. Both liver and brain show similar tendency to reduce number of high affinity receptors and their affinity to the hormone in rats fed a HP diet. After a HC diet the effects seem to be opposite. Animals show nearly the same number of receptors as in the control group but the affinity of insulin binding to liver but not brain plasma membranes is enhanced.


INTRODUCTION
Food consumed by animals is not only a source of components used by tissues for energy or as a building material, but also regulates secretion of many substances, including insulin, which is one of the most important anabolic hormones.Insulin mediates several important functions involved in cellular metabolism of proteins (Lewis et al., 1992), lipids (Susini et al., 1979) and carbohydrates (Lawrence et al, 1976).Action of insulin is regulated by the rate of its secretion and it is well known that fasting lowers insulin concentration in blood and sugars or amino acids evoke enhancement of its secretion from pancreas (Mackowiak, 1990;Steffens et al., 1984).However, effects caused by insulin in target tissues depend not only on its level in blood but a major role plays interaction of the hormone with membrane receptors, which transmit signals to the intracellular space.Some aspects of the food influence on insulin receptors were investigated by Okamoto et al. (1992) on rats fed a lipid-rich diet, and by Dardevet et al. (1991) who carried out experiments on liver of growing rats ISSN 1230-1388 © Institute of Animal Physiology and Nutrition after a protein-rich diet.On the other hand, changes of receptors were noticed also after fasting (Almira and Reddy, 1979).
This study was undertaken in order to put more light on the linkage of sugar and protein content in a fodder with a number and affinity of insulin receptors which can be regarded as markers of tissue activity.On the other hand, only scanty data on the brain insulin receptors are available and lots of questions concerning their role and action need further explanations (Unger et al, 1991a, b).

MATERIAL AND METHODS
Fifteen adult male Wistar rats, weighing about 360 g, were housed individually in standard room conditions with free access to food and water.Control group (five animals) was kept on Murigran diet (commercial food produced by Bacutil-Poland, in %: wheat bran -10, ground corn -17.5, ground wheat -20, ground oat -15, fish meal -8, skim -milk powder -12, casein -5.5, yeast powder -5, dried green forage -5, mineral and vitamin additives -2; metabolizable energy -12.8 MJ/kg of dry matter, crude protein -24%, crude fat -3.7%).This diet was supplemented with fish meal (60% crude protein, 7.5% crude fat) to reach final concentration of 40% protein (high-protein group, five animals) or with 25% of chemically pure starch (high-carbohydrate group, five animals).After two weeks of feeding experimental diets, rats were decapitated and tissues were taken for analyses.
Brain and liver plasma membranes were prepared according to the method of Havrankova et al. (1978).Briefly, tissues were homogenized in NaHC03 (1 mmol/1) and centrifuged at 600g for 30 min.Obtained supernatants were centrifuged at 20000g for 30 min.and pellets were resuspended in 1 mmol/1 NaHC03; this step was next repeated and the final suspensions, after protein determination by a method of Lowry et al. (1951), were centrifuged once more in the same manner.Obtained pellets of membranes were resuspended in the incubation buffer (Tris 40 mmol/1, pH 7.4, containing 0.2 g/1 BSA).All procedures mentioned above were carried out at 4°C.
In order to investigate insulin binding, 80 pg of 125 I-labelled porcine hormone (OPiDI Swierk, Poland) and varying amounts of unlabelled insulin (0.02-700 nmol/1) were incubated (16h at 4°C) with previously prepared membranes (final concentration of proteins -0.5 mg/ml for liver and 1.0 mg/mol for brain plasma membranes).The nonspecific binding of 125 I-insulin was determined at 10 /rniol/1 of unlabelled insulin and subsequently subtracted from each value.Finally, after total radioactivity had been measured, vials were centrifuged at 20 OOOg, (4°C, 8 min) and obtained pellets were used for counting.
Dissociation rate constant (Kd) and binding capacities (Bmax) for insulin were determined by the Scatchard's analysis method (Scatchard, 1949) using the TYPE OF DIETS -LIVER AND BRAIN INSULIN RECEPTORS 209 LIGAND-PC v.3.1 computer program (Munson and Rodbard, 1980).Binding potency of insulin was calculated as IC50 using the ALLFIT-PC v. 2.7 computer program (De Lean et al., 1978).
The results were interpreted according to one-way analysis of variance.Significance of differences between groups was calculated using multiple range test.

RESULTS
Obtained results indicate that both liver and brain plasma membranes in adult rats show ability to bind pork insulin.However, markedly higher specific insulin binding was observed for liver.Effects of increasing concentrations of unlabelled insulin on 125 I-insulin displacement in membranes of investigated tissues are shown in Figure 1.The data on the kinetics of the insulin binding are presented in form of Scatchard plot in Figure 2. Analysis of these typically curvilinear plots allows us to distinguish in liver and brain both-high (HAIR) and low (LAIR) affinity insulin receptors.Their number and characteristic features are summarized in Table 1.
Protein-rich diet diminishes significantly specific binding of insulin in liver and brain, whereas high-carbohydrate diet almost does not influence insulin action.Curves representing binding of 125 I-labelled insulin to brain or liver lie in the case of high-protein group markedly below other ones.Simultaneously, high-protein diet diminishes significantly by about 32% HAIR number and lowers their affinity by 22% in liver.In contrary, high-carbohydrate diet which practically does not change HAIR number, elevates their affinity by 43%.That effect is extremely visible while high-protein and high-carbohydrate groups are compared.In that case affinity of receptors in the last group is almost twice TYPE OF DIETS -LIVER AND BRAIN INSULIN RECEPTORS 211 higher.In brain diets cause similar changes.Animals kept on a high-protein food have reduced specific binding by 16% and lowered amount of HAIR by 21%.Also, affinity of HAIR is diminished by 25%, whereas after high-carbohydrate food it is unchanged.Insulin is a well characterized hormone which influences protein, carbohydrate and lipid metabolism.Because of the important function, there exist some mechanisms which control its action.One of them is regulation of insulin output from pancreas by many important ingredients of food.On the other hand, there are evidences that not only insulin secretion but also its action on target tissues can be regulated (Dardevet et al., 1991;Okamoto et al., 1992).The last phenomenon can take place under condition that number of receptors and/or their affinity to the hormone are changeable or some post-receptor events can be influenced by intermediary metabolites or insulin itself.In our previous investigations we stated that present in a diet hormone-like substances, called phytooestrogens, change receptor content and reactivity of liver, muscle and blood red cells to insulin (Nogowski et al., 1991a, b).Results presented here indicate that changes in basic food ingredients can also influence on the receptor content and affinity of liver and brain to the hormone.The carbohydrate-rich diet caused some enhancement of specific insulin binding both in liver and brain membranes.This effect seems to be not a result of increased quantity of binding places but their enhanced affinity.Simultaneously, excess of protein in diet caused lowering of HAIR level and their affinity to insulin in investigated tissues.The phenomenon of food influence on receptors was reported by Okamoto et al. (1992), who observed that a lipid-rich diet (36% fat) diminished the autophosphorylation of muscle and liver insulin receptors on tyrosine residues and caused their resistance to insulin.However, the authors stated no changes in the insulin binding, receptor number or affinity constant.Their data suggest that a high-fat diet causes a post-receptor defect.In other studies Dardevet et al. (1991) stated that a high-protein food decreased number of high affinity receptors in liver of growing rats.On the other hand, fasting increases both the quantity of binding sites and specific binding (Almira and Reddy, 1979).Our experiments confirmed lowering of HAIR number in liver after protein-rich diet and -in addition -we noticed that this same effect was also revealed in brain.Moreover, some effects evoked by this kind of diet are opposite to that observed after excess of carbohydrates which enhance association constant in liver.
The direction of changes was, in general, the same in liver and brain, however the last organ seems to be less sensitive.Brain membrane high affinity receptors show lower affinity to insulin than liver ones.It can be a result of differences in structure of receptors originating from various tissues.The brain receptor is a bit smaller molecule and this distinction arises from differences in N-linked glycosylation (Unger et al, 1991a;Gammeltoft et al., 1984).However, besides described differences in the characteristics of brain and liver membrane receptors, in the light of the present paper, it can be stated both their ability to changing in various circumstances and the regulatory effect of a diet on them.It seems to be very interesting especially referring to brain, which is usually considered not to be a target tissue for insulin.Badano blonowe receptory insulinowe wa^troby i mozgu szczurow zywionych pasza^ standardowq.(S), wysokoweglowodanowa^ (HC) i wysokobialkowa^ (HP).Zarowno wqtroba jak i mozg wykazuja^ podobnq tendency do redukcji receptorow o wysokim powinowactwie, a takze obnizania ich powinowactwa u zwierz^t zywionych pasza^ HP.Dieta HC wydaje si? wywierac dzialanie odwrotne.Zwierzeta majg.prawie taka.sama, ilosc receptorow jak w grupie kontrolnej, ale powinowactwo wigzania insuliny do blon watrobowych -lecz nie mogzowych -jest podniesione.