Insulin Binding Characteristics Research Articles

Insulin binding characteristics in canine muscle tissue: effects of the estrous cycle phases

Abstract: Hormonal fluctuations during the different estrous cycle are a well-recognized cause of insulin resistance in bitches, and little is known about insulin receptor binding or post-binding defects associated with insulin resistance in dogs. To evaluate insulin binding characteristics in muscle tissue of bitches during the estrous cycle, 17 owned bitches were used in the study (six in anestrus, five in estrus, and six in diestrus). An intravenous glucose tolerance test (IVGTT) was performed in all patients by means of injection of 1mL/kg of a glucose 50% solution (500mg/kg), with blood sample collection for glucose determination at 0, 3, 5, 7, 15, 30, 45 and 60 minutes after glucose infusion. Muscle samples, taken after spaying surgery, were immediately frozen in liquid nitrogen and then stored at -80 ºC until the membranes were prepared by sequential centrifugation after being homogenized. For binding studies, membranes were incubated in the presence of 20,000cpm of human 125I-insulin and in increasing concentrations of unlabeled human regular insulin for cold saturation. The IVGTT showed no differences among bitches during the estrous cycle regarding baseline glycemia or glycemic response after glucose infusion. Two insulin binding sites - high-affinity and low-affinity ones - were detected by Scatchard analysis, and significant statistical differences were observed in the dissociation constant (Kd1) and maximum binding capacity (Bmax1) of the high-affinity binding sites. The Kd1 for the anestrus group (6.54±2.77nM/mg of protein) was smaller (P<0.001) than for the estrus (28.54±6.94nM/mg of protein) and diestrus (15.56±3.88nM/mg of protein) groups. Bmax1 in the estrus (0.83±0.42nM/mg of protein) and diestrus (1.24±0.24nM/mg of protein) groups were also higher (P<0.001) than the values observed in anestrus (0.35±0.06nM/mg of protein). These results indicate modulation of insulin binding characteristics during different phases of the estrous cycle in dogs, showing that muscle insulin binding affinity for its receptor is reduced during estrus and diestrus. However, this poor hormone-receptor affinity is compensated for by a greater total binding capacity, once there is no difference in patients' glycemic response after an intravenous glucose load.

Insulin binding monitored by fluorescence correlation spectroscopy.

The characteristics of insulin binding to its receptors have been extensively studied by the radioligand binding assay. We used fluorescence correlation spectroscopy to determine the distribution of diffusion times and further novel data on the kinetics of insulin's binding to its receptor. Cultured human renal tubular cells (HRTC) were incubated with tetramethyl rhodamine labelled insulin (Rh-Ins) for 60 min. Fluorescence intensity fluctuations and autocorrelation functions for Rh-Ins, free in the incubation medium and bound to the cell membrane, were studied at single-molecule detection sensitivity in a 0.2 fL confocal volume. Measurements at the cell membrane revealed Rh-Ins binding with at least two diffusion components (diffusion times tauD1 = 0.8 ms, tauD2 = 20 ms) and corresponding weight fractions of y1 = 0.43 and y2 = 0.42. Specificity of the binding was shown by the dislocation of bound Rh-Ins when excess unlabelled insulin was added. Scatchard analysis showed a nonlinear plot, revealing two binding processes with different affinities (Kass approximately 2 x 10(10) M(-1) and approximately 1 x 10(9) M(-1), respectively). The fluorescence correlation spectroscopy results show two classes of binding sites with different affinities for insulin, or interactions between receptor sites consistent with negative cooperativity. This conclusion is in agreement with studies of insulin binding using radioligand binding assays. Because of its high sensitivity (single molecule detection), FCS, provides additional data allowing a more precise evaluation of the kinetics of ligand-receptor interactions at low expression levels in living cells.

Mechanisms of Insulin Action and Na+/H+ Exchanger in Adipocytes.

Characteristics of insulin binding to isolated rat adipocytes were studied by use of fluorescein isothiocyanate (FITC)-labeled insulin and flow cytometry, and were compared with those of insulin-induced uptake of 3H-2-deoxy-D-glucose (3H-2-DG). FITC-labeled insulin bound to the adipocytes during the initial incubation, but washing once with Hanks' buffer completely removed the bound insulin from the cells. Uptake of 3H-2-DG by the adipocytes was induced by insulin but ceased when the cells were washed. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na+/H+ exchanger inhibitor, suppressed both the insulin-induced 3H-2-DG uptake and insulin-induced translocation of glucose transporter type 4 (Glut-4) from the microsomal membranes to the plasma membrane of the cells. In the absence of insulin, when the Nay in the Hanks' buffer was replaced with Li+[Na+(-)Li+(+) Hanks' buffer], the level of 3H-2-DG uptake by the cells was significantly higher than in the presence of Na+. The increase in the uptake of 3H-2-DG induced by Li+ was similar to the increase induced by insulin, but phosphorylation of the insulin receptors was not detected by Western blotting. The increased 3H-2-DG uptake induced by the Na+(-)Li+(F) Hanks' buffer solution was abolished by adding EIPA. This result suggests that the Na+/H+ exchanger influences Li+-induced 3H-2-DG uptake. Based on these results, we conclude that the observed insulin-induced glucose uptake was tightly regulated by the Na+/H+ exchanger.

Mitogenic properties of insulin and insulin analogues mediated by the insulin receptor.

Insulin has traditionally been considered as a hormone essential for metabolic regulation, while the insulin-like growth factors (IGF-I and IGF-II) are postulated to be more specifically involved in growth regulation. The conventional wisdom is that they share each other's effects only at high concentrations, due to their weak affinity for the heterologous receptor. We discuss here the evidence that in the proper cellular context, insulin can be mitogenic at physiologic concentrations through its own receptor. We studied the insulin and IGF-I binding characteristics of a new model suitable for analysing insulin receptor mediated mitogenesis; that is, a T-cell lymphoma line that depends on insulin for growth, but is unresponsive to IGFs. The cells showed no specific binding of 125I-IGF-I and furthermore, no IGF-I receptor mRNA was detected by RNAse protection assay in the LB cells, in contrast with mouse brain and thymus. The cells bound at saturation about 3000 insulin molecules to receptors that had normal characteristics in terms of affinity, kinetics, pH dependence and negative co-operativity. A series of insulin analogues competed for 125I-insulin binding with relative potencies comparable to those observed in other insulin target cells. The full sequence of the insulin receptor cDNA was determined and found to be identical to the published sequence of the murine insulin receptor cDNA. The LB cell line is therefore an ideal model with which to investigate insulin mitogenic signalling without interference from the IGF-I receptor. Using this model, we have started approaching the molecular basis of insulin-induced mitogenesis, in particular the role of signalling kinetics in choosing between mitogenic and metabolic pathways.

Reduced Cell Attachment and Phosphorylation of Focal Adhesion Kinase Associated with Expression of a Mutant Insulin Receptor

Insulin signaling results in rapid changes to the cell cytoskeleton, and it has recently been shown that insulin stimulates the dephosphorylation of the cytoskeletal-associated tyrosine kinase, focal adhesion kinase (pp125(FAK)). We report here that mutation of two tryptic cleavage sites (Lys164 and Lys582 --> Asn; 2N) in the insulin receptor alpha-subunit results in a cell-line (CHO.2N-10) with altered morphology associated with an increase in cell size, a decrease in cell adhesiveness, and a decrease in pp125(FAK) tyrosine phosphorylation in the absence of insulin (45.2 +/- 9.7% compared to nontransfected Chinese hamster ovary (CHO) cells). In contrast to pp125(FAK), paxillin phosphorylation was similar in all cell lines despite lower levels (61.0 +/- 10.4% compared to CHO cells) of paxillin protein in CHO.2N-10 cells. We observed comparable protein levels of pp125(FAK) and the structural focal adhesion protein, vinculin, in all cell lines. Despite underphosphorylation of pp125(FAK) in the basal state, insulin stimulation of CHO.2N-10 cells still resulted in dephosphorylation of pp125(FAK). CHO.2N-10 and CHO.T (overexpress wild-type insulin receptor) cells have similar insulin binding characteristics, insulin-stimulated autokinase and peptide phosphorylation, and insulin-stimulated pp185/IRS-1 phosphorylation. Our results suggest that the insulin receptor may play an important role in cell-matrix interactions, such as modulating cell adhesion and inducing cell architecture changes.

Glycosylation of Asn397 or Asn418 is Required for Normal Insulin Receptor Biosynthesis and Processing

Two N-linked sites of glycosylation in the insulin receptor were examined for their contribution to insulin binding, tyrosine kinase activity, and receptor biosynthesis. Asn397 and Asn418 were replaced by Gln using site-directed mutagenesis either as single mutations, i.e., Q-397 and Q-418, or as a double mutation in which both sites were removed (Q-D). The mutations were transiently expressed in COS cells and the findings compared with cells that transiently expressed the wild-type human insulin receptor. Q-397 and Q-418 mutant insulin receptors had insulin-binding characteristics similar to the wild-type human insulin receptor, whereas no insulin-binding activity could be detected above the control level in cells transfected with Q-D. Flow cytometry with antibodies against the human insulin receptor indicated the presence of Q-397, Q-418, and wild-type human insulin receptors in the surface of COS cells and failed to demonstrate a Q-D receptor. Insulin-induced autophosphorylation was similar in Q-397, Q-418, and wild-type human insulin receptors as was their ability to phosphorylate an artificial substrate, poly Glu-Tyr (4:1). Our inability to detect Q-D receptors was not caused by a lack of Q-D mRNA. COS cells transfected with Q-D cDNA generated as much Q-D mRNA as the amount of wild-type human insulin receptor mRNA present in cells transfected with wild-type receptor cDNA. Finally, pulse-chase experiments with [35S]Met were able to detect 190,000-M(r) proreceptors and the alpha-subunits for Q-397, Q-418, and wild-type human insulin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Inability of insulin and insulinlike growth factor-1 to stimulate sugar or amino acid transport and thymidine incorporation in cultured myeloma cells

NS-1 mouse plasmacytoma cells were examined for their insulin and insulinlike growth factor-1 (IGF-1) binding characteristics and ability to produce peptide-dependent cellular effects. At concentrations of labelled insulin (i.e., 1.7 x 10(-10) M) or IGF-1 (i.e., 1.5 x 10(-10) M), NS-1 cells specifically bind 0.2 +/- 0.06 fmol insulin per 10(6) cells (n = 7), where little, if any, IGF-1 specific binding was observed (0.02 +/- 0.01 fmol/10(6) cells) (n = 3). Additionally, the data indicate that the total number of insulin binding sites per cell was 3200 +/- 390 (n = 3). Insulin was employed at various concentrations (6.7-667 nM) and failed to stimulate either sugar or amino acid transport. Insulin at low concentrations (i.e., 6.7 or 67 nM) did not stimulate DNA synthesis, yet a small but significant increase was observed at a concentration of 667 nM insulin. IGF-1 did not stimulate DNA synthesis at all concentrations employed (1.4-143 nM). In summary, there exists a small but significant number of insulin receptors, little insulin-stimulated DNA synthesis, and no apparent insulin stimulation of sugar or amino acid transport. Also, since there is no significant IGF-1 binding and no IGF-1 stimulation of DNA synthesis, these findings indicate that this cell line might be a good candidate for the study of insulin receptor function as a transfection recipient of insulin receptor genes.

Regulation of adipose tissue metabolism during pregnancy and lactation in the ewe: the role of insulin1

This study was conducted to examine the effect of insulin on lipid metabolism of adipocytes during pregnancy and lactation in ewes. During the first 3 mo of pregnancy, metabolism of adipocytes from omental adipose tissue was characterized by a high rate of de novo lipogenesis (90 to 125 nmol of acetate incorporated into lipids.2 h-1.10(6) cells-1) and a 38% reduction in response to beta-lipolytic stimulus (isoproterenol 10(-6) M). Simultaneously, there was a rise in the number of high-affinity insulin receptors (Kd = .2 nM), and insulin binding characteristics showed a decrease in the negative cooperativity phenomenon. Moreover, lipogenesis stimulated by insulin (1 mU/ml) increased in comparison with observations in nonpregnant ewes. The last third of pregnancy and early lactation were characterized by a marked fall in lipogenesis and a simultaneous increase in isoproterenol-stimulated lipolysis. During lactation, the number of total insulin receptors was decreased by 62% and insulin stimulation of lipogenesis became inefficient. Results suggest that insulin plays a direct role in adipose tissue metabolism during pregnancy.

Tyrosine-Kinase Defect of the Insulin Receptor in Cultured Fibroblasts from Patients with Lipoatropic Diabetes*

Postbinding defects in insulin action were described previously in cultured fibroblasts from six patients with lipoatropic diabetes. To define the contribution of the insulin receptor tyrosine kinase in these defects, we studied autophosphorylation and kinase activity of lectin purified receptors from these six patients and six normal cell lines. The patients' insulin receptors, prepared by precipitation with polyethylene glycol, had normal insulin binding characteristics and autophosphorylation properties, but a 56% decrease in the tyrosine kinase activity toward an exogenous substrate. To identify more subtle qualitative defects in autophosphorylation, insulin receptors were sequentially immunoprecipitated and analyzed for their phosphoaminoacid content. The phosphorylated receptors precipitated with an antiphosphotyrosine antibody contained labeled phosphotyrosine, whereas those in the supernatant, when further precipitated with an antireceptor antibody, contained only phosphoserine. Under these conditions, the insulin-stimulated autophosphorylation of tyrosine was significantly decreased by 54% in the patient receptors compared to normal subjects' receptors. In addition, insulin-like growth factor-I stimulation of autophosphorylation of its receptor was reduced by 59% in the patients' cells compared to those from normal subjects. We conclude that fibroblasts from patients with lipoatropic diabetes have defects in the tyrosine kinase activity of their insulin and their insulin-like growth factor-I receptors that might give rise to the in vitro hormone resistance and be related to the in vivo hormone resistance that occurs in these patients.

Reduced insulin removal and erythrocyte insulin binding in obese children.

To study the relationship between childhood obesity, weight loss, hyperinsulinaemia and the erythrocyte insulin receptor, we measured the plasma concentrations of immunoreactive insulin (IRI) and C-peptide and the binding of 125I-insulin to erythrocytes in 12 obese children with a mean age +/- SD of 11.4 +/- 2.5 years and a mean relative weight score +/- SD of 4.8 +/- 1.4 and 12 age-matched normal-weight children. Eight obese children were re-evaluated after 1 year's participation in a weight reduction programme. The obese children had higher fasting plasma concentrations of IRI (P less than 0.01) and C-peptide (P less than 0.05) and a lower C-peptide to IRI molar ratio (P less than 0.01) than the normal-weight children. The obese children had in addition a reduced erythrocyte insulin binding (P less than 0.05 or less) over the physiological range of circulating insulin concentration. There was a negative correlation (r = -0.60; P less than 0.01) between the insulin tracer binding and the relative weight. The weight reduction programme resulted in a decrease of 1.0 SD (P less than 0.05) in the mean relative weight score. At the end of the therapy the obese children had lower fasting blood glucose levels (P less than 0.05) and lower plasma IRI concentrations at 90 min (P less than 0.05) after an oral glucose load than at the onset of therapy. There were no significant differences between the insulin binding characteristics at the commencement and at the end of the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective insulin receptor tyrosine kinase in human skeletal muscle in obesity and type 2 (non-insulin-dependent) diabetes mellitus.

The tyrosine kinase activity of the insulin receptor was investigated in skeletal muscle biopsies from insulin-resistant males with obesity or with Type 2 (non-insulin-dependent) diabetic males who were lean or overweight. The kinase activity of the receptor from all three groups of insulin-resistant subjects was 40% less when compared to the activity of lean control subjects. This alteration was present in the absence of changes in the level of the insulin receptor on its insulin binding characteristics. We conclude that the tyrosine kinase activity of the skeletal muscle insulin receptor is defective in obesity and Type 2 diabetes, and that this alteration contributes to the insulin-resistant characteristics of both disorders.

Insulin binding and biological activities in the FRTL-5 rat thyroid cell line

A cloned rat thyroid cell line (FRTL-5) was examined for both insulin binding and responsiveness. The characteristics of insulin binding to thyroid cells were similar to those observed in typical insulin target cells. The 125I-insulin binding was time and temperature dependent and Scatchard analysis suggested the presence of two major binding sites with high and low affinity constant (Kd = 1.4 × 10 −10 mol/L and 1.5 × 10 −9 mol/L, respectively). 125I-insulin was also internalized and degraded in a temperature-dependent manner. IGF 1 was weakly effective in completing 125I-insulin binding to FRTL-5 cells (57% inhibition at 333 nmol/L), whereas noninsulin-related peptide hormones were ineffective. Exposure of FRTL-5 cells to insulin stimulated both methyl-aminoisobutyric acid (M-AIB) and 2-deoxy-D-glucose (2DG) transport. These effects were evident at 10 −9 mol/L and maximal at 10 −7 mol/L insulin. Maximal stimulation was three- to four-fold over basal value for both M-AIB and 2DG transport. These data suggest that insulin specifically binds to FRTL-5 cells and regulates different biological functions of this thyroid cell line.

Effects of chronic administration of vanadate to the rat on the sensitivity of glycolysis and glycogen synthesis in skeletal muscle to insulin

Male Wistar rats were given sodium orthovanadate in their drinking water for at least 14 days. This treatment increased the hypoglycaemic effect of intravenously administered insulin and increased the sensitivity of isolated soleus muscle strips to insulin with respect to both glycolytic and glycogen synthetic rates. This effect of chronic vanadate administration was shown not to be a consequence of a change in the insulin binding characteristics of soleus muscle. It is suggested that these changes may be brought about by the interaction of vanadate with insulin-mediated alterations in tyrosine kinase/phosphotyrosyi phosphatase activities.

Stimulation of Na +Ca 2+ exchange in heart sarcolemma by insulin

Insulin was found to stimulate Na +-dependent Ca 2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulations (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na +-dependent Ca 2+ uptake, ATP-dependent Ca 2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca 2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na +K +-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 × 10 9 M −1 and 4.4 × 10 8 M −1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca 2+ transport systems.

A thiol-sensitive degradative process of liver uncouples autophosphorylation of the insulin receptor from insulin binding.

Insulin receptors derived from highly purified rat liver plasma membranes and Golgi membranes showed differences in insulin-mediated receptor autophosphorylation, even though their insulin-binding characteristics were similar. This difference was related to the generation of a Mr-84,000 fragment of the Mr-90,000 beta subunit of the plasma-membrane receptor, a fragment that was not present in the receptor from Golgi membranes, in the absence of a change in the insulin-binding alpha subunit. When autophosphorylation activity was based on insulin binding, the activity of the plasma-membrane-derived insulin receptor was decreased to 25-30% that of the Golgi-derived receptor. Endoglycosidase F digestion produced changes in the Mr values for both species, but they were not converted into a single subunit, thereby suggesting differences in the protein component of the two subunits. Although the proteinase inhibitors phenylmethanesulphonyl fluoride, ovomucoid and aprotinin failed to block the formation of the Mr-84,000 fragment, the presence of iodoacetamide or EDTA during liver homogenization markedly inhibited fragment generation and allowed the plasma-membrane insulin receptor to retain an autophosphorylation activity comparable with that present in insulin receptors from Golgi membranes. Thus a thiol-sensitive, cation-dependent, degrading activity has been identified that can uncouple the insulin-binding activity of the plasma-membrane insulin receptor from its tyrosine kinase activity.

Insulin binding and insulin action in cultured fibroblasts: significant differences between a phosphoglucose isomerase-deficient mutant and the parental strain.

The interrelationships of hexose feeding and insulin action were studied in the Chinese hamster fibroblast cell lines 023 and DS-7. The latter, derived from 023 and deficient in phosphoglucose isomerase, has been used to map the metabolic requirements for aldohexose-mediated down-regulation or "curbing" of hexose transport. We have characterized insulin binding and the response to insulin in both cell lines to determine if the insulin-mediated stimulation of transport is similarly dependent on hexose metabolism. DS-7 cells exhibited 5-6 times as many high-affinity insulin binding sites as the parental strain. Apart from this difference, 023 and DS-7 cells showed comparable insulin binding characteristics, which are similar to those observed in other cell types. Insulin at a concentration of 1 microgram/ml (167 nM) was found to stimulate 3-O-methylglucose uptake by approximately equal to 50% in glucose-fed cells of both lines. In neither line did glucose starving significantly alter insulin binding or the insulin-induced stimulation of transport. Feeding with mannose or fructose was found to increase both parameters in 023 cells but had no effect on DS-7 cells. The increase in hexose uptake with the administration of insulin or with glucose starving was shown to be due to an increase in Vmax. Our studies suggest that insulin binding and effect are not regulated by hexose metabolism in the same manner as in the curbing process and insulin induces the recruitment of a transporter pool that is insensitive to hexose curbing.

Studies of the human liver insulin receptor in noninsulin-dependent diabetes mellitus.

The insulin binding characteristics and the structural components of the insulin receptor were studied in the purified liver plasma membranes from seven patients with noninsulin-dependent diabetes (NIDDM) and seven control subjects. In comparison to the controls, diabetic subjects had a 65% reduction in plasma insulin levels in response to an oral glucose load. Specific insulin binding by liver membranes from diabetic patients was, however, twofold greater than the binding activity by membranes from control subjects. This alteration resulted largely from an increase in the number of insulin receptors and a modest increase in receptor binding affinity. Holo (nonreduced) receptor species of similar molecular weights were detected by an affinity labeling technique in the two membrane preparations and sulfhydryl reduction demonstrated an insulin binding subunit of 125,000 mol wt. Overall, these results show that the hepatic insulin resistance of NIDDM cannot be explained by a deficiency in insulin binding.

Effect of fasting and dexamethasone on binding characteristics of canine erythrocyte insulin receptors

Insulin binding characteristics of canine erythrocyte insulin receptors were studied before and after a 72-hour fast, and one and three days following glucocorticoid (dexamethasone) administration. The 72-hour fast tended to increase maximum insulin binding, but no significant differences were found. The administration of dexamethasone resulted in an increased maximum binding of insulin to its receptors which, at day 1, was due to an increase in receptor concentration, and at day 3, to an increased insulin binding affinity of the receptor. These data suggest that the erythrocyte insulin receptor may be useful in clinical and experimental studies in the dog.

Erythrocyte Insulin Binding in Preterm Newborn Infants

To characterize the erythrocyte insulin receptor in newborn infants we studied the binding of 125I-insulin to the erythrocytes from 42 preterm infants (14 at birth, 14 aged 2-7 days, and 14 aged 8-16 days) with a mean gestational age of 34.1 wk, and from 32 term infants (16 at birth and 16 aged 2-7 days). The insulin binding to cord blood erythrocytes from preterm infants was significantly higher than that of cord blood cells from term infants and to postnatal cells from preterm as well as term infants. The erythrocytes from preterm infants aged 2-7 days bound more insulin than cells from preterm infants aged 8-16 days. The maximum insulin binding (specific insulin binding at tracer concentration of insulin) correlated negatively with the gestational age both at birth and over the 1st postnatal wk. In the preterm infants there was a strong negative correlation between the maximum insulin binding and postnatal age. The enhanced insulin binding to cord blood erythrocytes from preterm infants was due to both an increased receptor concentration and a high affinity for insulin. The increased affinity persisted over the 1st wk of life. In preterm infants older than 1 wk the insulin binding characteristics were basically similar to those in term newborn infants. In all infants studied the receptor concentration seemed to be postnatal age dependent while the receptor affinity was gestational age dependent. No correlation was found between the insulin binding data and the plasma concentrations of immunoreactive insulin or C-peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth.

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.