Hypothalamic CRF immunoreactivity in genetically hypothyroid (hyt/hyt) mice

The induction of hypothyroidism in young rats by feeding thiouracil to their mothers during pregnancy has been shown to depress hypothalamic content of bioactive and immunoactive corticotropin-releasing factor (CRF). The present study was done to determine whether genetically hypothyroid young mice (hyt/hyt) born to euthyroid mothers (+/hyt) exhibited a similar depression in hypothalamic CRF immunoreactivity. Young euthyroid and hypothyroid littermate mice were examined by radioimmunoassay for hypothalamic CRF content at 15, 20, 25, or 30 days of age. Mean CRF content was depressed insignificantly (to about 80% of normal) by hypothyroidism, at 15-25 days of age. However, after weaning by the mother, 30-day-old hypothyroid pups demonstrated significantly depressed hypothalamic CRF levels (71%). It is suggested that maternal factors may be assisting in the maintenance of hypothalamic CRF until after weaning. Furthermore, genetic hypothyroidism does not appear to have nearly as marked an influence as thiouracil feeding on hypothalamic CRF levels.     

Effect of thyroid hormone and sex status on epidermal growth factor concentrations in the submandibular gland of a congenitally hypothyroid mouse model

Studies were performed to explore the role of thyroid hormone and sex status on epidermal growth factor concentrations in the submandibular gland of a congenitally hypothyroid mouse model designated hyt/hyt. The animals were studied at 20, 30 and 40 days of postnatal age. The euthyroid animals were homozygous or heterozygous for the hypothyroid gene. The homozygous euthyroid animals displayed a pattern of submandibular gland epidermal growth factor concentrations similar to those previously described in other mouse species and showed the expected sex differences. The hypothyroid animals had measurable but very low submandibular gland epidermal growth factor concentrations without sexual dimorphism. Serum thyroxine concentrations in the heterozygotes were comparable to those in the homozygous euthyroid animals, yet the animals had a delayed increase in epidermal growth factor concentrations combined with a later expression of female-male differences. The timing of the sex differences in submandibular gland epidermal growth factor concentrations followed a pattern similar to that seen for the timing of the first litter in these three genetically distinct groups. This infers the timing of the onset of puberty and suggests a role of androgens in the changes seen in epidermal growth factor concentrations. We conclude that thyroid hormone and sex status in this mouse model influence the pattern and concentrations of submandibular gland epidermal growth factor concentrations.     

Nerve growth factor concentration in a congenitally hypothyroid mouse model (hyt/hyt) and its responsivity to thyroxine treatment

Earlier our laboratory reported the ontogenic profiles of serum thyroxine (T4) and triiodothyronine (T3) concentrations and nerve growth factor levels in the submandibular gland of Swiss-Webster mice. Further, we demonstrated a responsivity of submandibular gland-nerve growth factor concentrations to exogenously administered T4. To further our understanding of the interactions between thyroid hormones and submandibular gland-nerve growth factor we utilized a congenitally hypothyroid mouse model to examine submandibular gland-nerve growth factor in euthyroid, hypothyroid and hypothyroid-T4 replaced mouse pups. Serum T4 values in the congenitally hypothyroid (hyt/hyt) mice were unmeasurable and their growth in body weight, their incisor eruption, and their eyelid opening were significant delayed. Submandibular gland/body weight ratios were significantly reduced relative to control or heterozygous (+/hyt) animals through 40 days. The increase in submandibular gland-nerve growth factor concentrations observed in normal animals before 20 days was delayed to 35 days in the (hyt/hyt) animals. T4 treatment of (hyt/hyt) animals from 11 or 18 days of age significantly increased mean 40 day submandibular gland/body weight ratios and submandibular gland-nerve growth factor concentrations. However, the 40 day submandibular gland-nerve growth factor levels in treated animals remained significantly below the level of control euthyroid mice. Thus, the possibility of critical time of thyroid hormone replacement for normal submandibular gland maturation has not been shown but must be further explored in this model.     

Impaired serum thyrotropin response to hypothyroidism in mice with disruption of neuromedin B receptor

Neuromedin B (NB), a neuropeptide highly concentrated in pituitary, has been proposed to be an inhibitor of thyrotropin (TSH) secretion. Previous study showed that mice with disruption of neuromedin B receptor (NBR-KO) have higher TSH release in response to thyrotropin-releasing hormone (TRH), although TSH seems to have decreased bioactivity. Here we examined in NBR-KO mice the response of TSH to thyroid hormone (TH) deprivation, obtained by methimazole treatment, or excess, obtained by acute and chronic TH administration. In response to hypothyroidism NBR-KO mice exhibited a lower magnitude increase in serum TSH compared to wild-type (WT) mice (1.7 vs. 3.3-times increase compared to euthyroid values, respectively, P<0.001). One hour after a single T4 injection (0.4 microg/100 g BW), WT and NBR-KO hypothyroid mice presented similar degree of serum TSH reduction (54%, P<0.05). However, 3 h after T4 administration, WT mice presented serum TSH similar to hypothyroid baseline, while NBR-KO mice still had decreased serum TSH (30% reduced in comparison to hypothyroid baseline P<0.05). T3 treatment of euthyroid mice for 21 days, with progressively increasing doses, significantly reduced serum TSH similarly in WT and NBR-KO mice. Also, serum T4 exhibited the same degree of suppression in WT and NBR-KO. In conclusion, disruption of neuromedin B receptor did not interfere with the sensitivity of thyroid hormone-mediated suppression of TSH release, but impaired the ability of thyrotroph to increase serum TSH in hypothyroidism, which highlights the importance of NB in modulating the set point of the hypothalamus-pituitary-thyroid axis at hypothyroidism.     

Effects of thyroid status and fasting on hepatic metabolism of apolipoprotein A-I

Metabolism of apolipoprotein (apo)A-I was studied in normal and chow-fed hyperthyroid rats, in 24-h fasted untreated male rats, and in rats after thyroparathyroidectomy (TXPTX). Rats were made hyperthyroid by administration of T3 (9.6 micrograms/day) or T4 (30 micrograms/day) with an Alzet osmotic minipump. Hyperthyroidism produced a similar two- to threefold elevation in plasma levels of apoA-I in male or female animals. During treatment with T3, plasma levels of T3 ranged from 200 to 400 ng/dl and did not correlate with plasma apoA-I levels. The net mass secretion and synthesis ([3H]leucine incorporation) of apoA-I by perfused livers from male hyperthyroid rats was elevated, while secretion of albumin was not different than that of euthyroid rats. Furthermore, the incorporation of [3H]leucine into total perfusate and hepatic protein was not altered by hyperthyroidism. The effect of thyroid hormone on apoA-I synthesis, therefore, does not appear to be a general effect on protein synthesis. After longer periods of treatment (28 days) with T3 (9.6 micrograms/day), hepatic apoA-I production decreased from that observed after 7 or 14 days of treatment, yet plasma apoA-I concentrations remained elevated. Plasma T3 decreased from 100 ng/dl to 40 ng/dl, in the hypothyroid rat resulting from TXPTX, but the plasma concentration of apoA-I did not change during the 2-week experimental period. The net secretion of apoA-I by livers from hypothyroid animals was depressed and albumin was uneffected compared to the euthyroid. Overnight fasting of euthyroid rats did not alter hepatic apoA-I secretion or plasma apoA-I levels, although under fasting conditions we had reported that hepatic output of apoB and E of VLDL is depressed. The addition of oleic acid to the perfusion medium, sufficient to stimulate VLDL production, did not affect net hepatic secretion of apoA-I by livers from euthyroid, hyperthyroid, or hypothyroid rats. In summary, hepatic synthesis of apoA-I appears to be controlled independently of other apo-lipoproteins and secretory proteins (albumin). Hepatic apoA-I synthesis is sensitive to thyroid status, increased in the hyperthyroid and decreased in the hypothyroid state. The specific stimulation of hepatic synthesis and secretion of apoA-I in the hyperthyroid state, however, tends to normalize over an extended period, perhaps from compensatory effects of a hormonal nature.     

Excretion of water, sodium, and potassium during the compensatory renal hypertrophy in hypothyroid rats]

Renal compensatory hypertrophy is studied in age matched euthyroid and radiothyroidectomized female rats. 7 days after uninephrectomy, the hypertrophy of the remaining kidney is equally small in both groups. But 60 days after this operation, the hypothyroid animals show only a 12% increase in the wet weight of the remaining kidney whereas the euthyroid controls increase this weight by 21%. The excretion of water, Na and K are determined in the urine excreted in 5 h after a small water load. The results are related to 1 gram of kidney wet weight. These outputs increase in all animals after uninephrectomy. They are significantly higher in the hypothyroid rats than in the euthyroid controls as well before than 60 days after uninephrectomy. The reduction in tubular Na reabsorption found in the hypothyroid rat may account for the impairment of compensatory renal hypertrophy in hypothyroidism.     

Regulation of uterine epidermal growth factor (EGF) receptors by estrogen in the mature rat and during the estrous cycle

Previous work has shown that the immature rat uterus contains epidermal growth factor (EGF) receptors and that tissue levels of this receptor are increased by the administration of exogenous estrogens. This study was undertaken to determine if estrogen administration also elevated EGF receptor levels in the mature animal and if the growth factor receptor levels varied in concert with endogenous estrogens throughout the estrous cycle. In the mature, castrate rat administration of estradiol, but not non-estrogenic steroids, causes a 2-3-fold elevation of uterine EGF receptors as judged by ligand binding. This increase is maximum in 18 h and is due to an increase in the number of binding sites. In cycling animals EGF receptor levels are low at metestrus, rise at diestrus, reach a maximum (approximately twice metestrus values) at proestrus, and then return at estrus to metestrus levels. These changes in EGF receptor levels parallel changes in plasma estrogens and occupied nuclear estrogen receptor reported by other workers. These results indicate that uterine EGF receptors are increased by exogenous estrogens in both mature and immature animals, and support a physiological role for estrogens in the regulation of this growth factor receptor.     

Thyroid hormone regulates alpha and alpha + isoforms of Na,K-ATPase during development in neonatal rat brain

The brain contains two molecular forms of Na,K-ATPase designated alpha found in non-neuronal cells and neuronal soma and alpha + found in axolemma. Previously we have shown that the abundance of both forms (determined by immunoblots) as well as Na,K-ATPase activity increases 10-fold between 4 days before and 20 days after birth (Schmitt, C. A., and McDonough, A. A. (1986) J. Biol. Chem. 261, 10439-10444). Hypothyroidism in neonates blunts these increases. Neonatal, but not adult brain Na,K-ATPase is thyroid hormone (triiodothyronine, T3) responsive. This study defines the period during which brain Na,K-ATPase responds to T3. The start of the critical period was defined by comparing Na,K-ATPase activity and alpha and alpha + abundance in hypothyroid and euthyroid neonates (birth to 30 days of age). For all parameters, euthyroid was significantly higher by 15 days of age. The end of the critical period was defined by dosing hypothyroid neonates with T3 daily (0.1 micrograms/g body weight) beginning at increasing days of age, and sacrificing all at 30 days then assaying enzyme activity and abundance. Those starting T3 treatment on or before day 19 were restored to euthyroid levels of Na,K-ATPase activity and abundance, while those starting T3 treatment on or after day 22 remained at hypothyroid levels of enzyme activity and abundance. We conclude that brain Na,K-ATPase alpha and alpha + isoforms are sensitive to T3 by as late as 15 days of age and that the period of thyroid hormone responsiveness is over by 22 days.     

Effect of hypothyroidism on the cytosolic free Ca2+ concentration in rat hepatocytes during rest and following stimulation by noradrenaline or vasopressin

The mean resting concentration of cytosolic free Ca2+ [( Ca2+]i) in parenchymal liver cells, as determined with the intracellular Ca2+ indicator quin2, was lowered by about 30% in hypothyroidism (0.17 microM vs. 0.27 microM in normal cells). The [Ca2+]i level in hypothyroid cells at 10 s following stimulation by noradrenaline (1 microM) was about 64% lower than in normal cells (0.33 microM vs. 1.0 microM). The response to noradrenaline in hypothyroid cells was slower in onset (significant at 5 s vs. 3 s in euthyroid cells), and the maximum of the initial [Ca2+]i increase was reached later (14 s vs. 8 s in normal cells). In hypothyroid hepatocytes the initial increase was followed by a slow but prolonged secondary increase in [Ca2+]i. With vasopressin similar results were found. Chelation of extracellular Ca2+ with EGTA immediately prior to stimulation had no effect on the initial [Ca2+]i increase. Treatment with T3 in vivo (0.5 micrograms/100 g body weight daily during 3 days) completely restored the basal and stimulated [Ca2+]i in hypothyroid cells. The half-maximally effective dose of noradrenaline was the same in euthyroid and hypothyroid liver cells (1.8 X 10(-7) M). Hypothyroidism had no significant effect on the number of alpha 1-receptors determined by [3H]prazosin labeling in crude homogenate fractions, while the Kd for [3H]prazosin was 21% lower than in the euthyroid group. These results show that thyroid hormone has a general stimulating effect on intracellular Ca2+ mobilization by Ca2+-mobilizing hormones, probably at a site distal to the binding of the agonist to its receptor. The results also support our idea that thyroid hormone may control metabolism during rest and activation, at least partially, by altering Ca2+ homeostasis.     

Role of thyroid hormones in the modification of diet-induced thermogenesis by propranolol

The effect of thyroid hormones on the reduction in resting metabolic rate by the beta-blocker propranolol has been investigated in young pigs. Oxygen consumption was measured 12 to 20 hr after the last meal in euthyroid and hypothyroid animals on a high or low level of energy intake. The increase in resting metabolic rate in animals on the high energy intake was reduced by propranolol in the euthyroid controls but not in those animals made hypothyroid by methimazole. Thus at least part of the action of propranolol to decrease diet-induced thermogenesis depends on its interaction with thyroid hormones.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an increase in protein kinases growth hepatic associated with epidermal factor receptor in the plasma membrane

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), administered to male rats at a single intraperitoneal (IP) injection dose of 25 μg/kg causes down-regulation of epidermal growth factor (EGF) receptor in the plasma membrane of rat liver which starts after two days and continues throughout the experimental period (20 days). Using monoclonal antibody to EGF receptor, it was determined that TCDD-caused EFG receptor down-regulation in the rat liver was accompanied by increased protein kinase activity. Such an increase in the protein kinase activity involves, at least in part, an activation of protein tyrosine kinase. Examination of serum samples from control and treated rats revealed no detectable difference in the level of EGF itself or EGF receptor-reacting substances (eg, hormones and other growth factors). In vivo TCDD caused early eye opening and tooth eruption and poor body weight gain and hair growth in mouse neonates similar to those observed with exogenously administered EGE The results indicate that such EGF receptor–mediated effect of TCDD has some toxocilogical significance in vivo. Although TCDD causes significant reduction in [¹²⁵I]-EGF binding in the hepatic plasma membrane in susceptible strains of mice, it has only modest effects in tolerant strains. The results are consistent with the idea that the action of TCDD on the EGF receptor is mediated through the cytosoliclnuclear TCDD receptor, which is known to be regulated by the Ah locus.     

In vivo stimulation of rat pineal type II thyroxine 5'-deiodinase activity by either norepinephrine or isoproterenol

Herein we show, for the first time, a very marked increase in thyroxine 5'-deiodinase (5'-D) activity in rats injected with norepinephrine (NE) and desmethylimipramine, a drug which inhibits NE uptake by nerve terminals. The response to NE was greater in pineals collected from hypothyroid animals than in glands from euthyroid animals. NE was more effective in stimulating pineal 5'-D than was isoproterenol, suggesting that, in addition to beta-adrenergic receptors, alpha-adrenergic receptors might be involved in the 5'-D activation. However, phenylephrine, an alpha-adrenergic agonist, did not potentiate the effect of isoproterenol on pineal 5'-D activity. The nocturnal increase in pineal 5'-D activity was completely abolished by propranolol, a beta-adrenergic receptor blocker, while prazosin, an alpha-adrenergic receptor blocker, had minimal effect. These results show that the role of alpha-receptors in promoting the NE-mediated rise in rat pineal 5'-D activity is minor in contrast to the role of beta-adrenergic receptors.     

Regulation of epidermal growth factor receptor number and phosphorylation by fasting in rat liver

The binding of 125I-epidermal growth factor (EGF) to microsomal membrane preparations from the livers of rats fasted for 72 h or fed control or high carbohydrate diets was examined to determine whether alterations in nutrient intake could affect the EGF receptor system. Fasted rats had 40-50% less membrane binding than did control or carbohydrate-fed rats. Scatchard analysis of the binding data indicated that the decrease in EGF binding in fasted rats was due to a decrease in receptor number with no change in receptor affinity. Cross-linking of 125I-EGF to EGF receptors with disuccinimidyl suberate revealed specific binding of a Mr 170,000 protein, which was diminished by approximately 75% in fasting, and a Mr = 150,000 protein, which accounted for 40-50% of the total labeling in the control and carbohydrate-fed rats and which was relatively unchanged by fasting. The sum of the labeling of the 2 bands was reduced by approximately 40% in fasting and is consistent with the reduction in EGF binding detected by Scatchard analysis. EGF stimulated a 1.5-3-fold increase in 32P incorporation into one major protein of 170 kDa in all 3 groups. Basal and EGF-stimulated autophosphorylation of 170 kDa, when normalized for protein, was 75% lower in membranes from fasted animals, compared to those from control or carbohydrate-fed rats. The comparable reduction of 125I-EGF binding to, and 32P incorporation into, the 170-kDa EGF receptor protein suggested that kinase activity/receptor was unaffected by fasting. Moreover, EGF receptor kinase activity in the 3 groups was comparable for an exogenous substrate, as judged by equal basal and EGF-stimulated phosphorylation of Val5-angiotensin II, when normalized for total EGF-binding capacity. These results suggest that fasting regulates EGF receptor kinase activity primarily by regulation of the number of hepatic EGF receptors. The possibility exists that some in vivo effects of fasting may be mediated by a reduction in EGF receptor levels.     

Thyroid hormone-, carbohydrate, and age-dependent regulation of a methylation site in the hepatic S14 gene

The rat hepatic S14 gene has served as a model of thyroid hormone regulation of gene expression. Earlier studies of the S14-containing chromatin region demonstrated that a cytosine residue at position 625 (C-625) in the 3' untranslated exon was hypermethylated in hepatic DNA derived from hypothyroid animals. This observation was consistent with the markedly reduced level of expression of the S14 gene in these rats. The current studies have extended these observations to groups of rats in various thyroidal states. By using the restriction enzyme Hhal, the percent demethylation of this site was quantitated (hypothyroid, 9.3%; euthyroid, 19.2%; hyperthyroid, 66.6%). Moreover, the level of methylation was shown to be reversible as the thyroidal state was altered. Our data also indicate that these changes are probably independent of de novo DNA synthesis. Kinetic studies of the demethylation of this cytosine residue after T3 administration showed no change for at least 1 day and maximal change after about 4 days. This contrasts with the significant rise in S14 mRNA evident within 30 min and suggests that demethylation plays no role in the acute induction of this gene by T3. Carbohydrate feeding, another stimulus of S14 expression, similarly caused the demethylation of this cytosine residue. Earlier studies had demonstrated that mRNA S14 expression was not detectable in rat pups before about 20 days of age and continued to rise through the first year of life. Consistent with those findings, S-14 C-625 was fully methylated up to 15 days of age. Progressive demethylation then occurred up to 12 months of age. These results indicate that increased demethylation of a specific site in the 3' untranslated region of the S14 gene, possibly resulting from augmented excision repair processes, is correlated with increased expression of the gene.     

Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number. Implications for existence of cryptic EGF receptors

Treatment of Swiss mouse 3T3 cells and human epidermoid carcinoma A431 cells with protamine at 37 degrees C increased the 125I-epidermal growth factor (EGF) binding activity at 4 degrees C. The effect of protamine on the increase of 125I-EGF binding activity appeared to be time, temperature, and dose dependent. This up-modulation of 125I-EGF binding by protamine correlated with protamine enhancement of EGF-stimulated mitogenesis, with respect to the magnitude of the effect and the dose response curves. Scatchard plot analyses indicated that protamine induced an increase in numbers of both high and low affinity EGF receptors without affecting their affinities. Protamine also increased functionally active EGF receptors in plasma membranes and solubilized membranes. This was evidenced by Scatchard plot analyses and by a protamine-induced increase of 125I-EGF-EGF receptor complex and an increase in EGF-stimulated phosphorylation of the EGF receptor. Combined with column chromatography of the solubilized EGF receptor on protamine-agarose gel, these results suggest that protamine may increase the EGF receptor number by directly activating cryptic EGF receptors in the plasma membrane.     

Thyroid hormone regulates tubulin expression in mammalian liver. Effects of deleting thyroid hormone receptor-alpha or -beta

Microtubules are made from polymers of alpha/beta dimers. We have observed in rat liver that, on the first day after birth, alpha-subunit is relatively high and beta-subunit low with respect to adult values. In the hypothyroid neonate, both subunits were found to be low, therefore indicating that thyroid hormone (TH) regulates these developmental changes. TH was also found to activate tubulin expression in adult liver, especially beta-subunit. To investigate the role of TH receptors (TRs) in tubulin expression, we analyzed mice lacking TRalpha or TRbeta compared with the wild type in both normal and TH-deprived adult animals. The results suggest that, in vivo, beta-tubulin protein expression in the liver is primarily under TRbeta positive control. In euthyroid mice lacking TRbeta, beta-tubulin expression was low. However, in the corresponding hypothyroid animals, it was found increased, therefore suggesting that the unliganded TRalpha might also upregulate beta-tubulin expression. Accordingly, TH administration to hypothyroid TRbeta-deprived mice reduced their high beta-tubulin expression. In parallel, the relatively high messenger level observed with these hypothyroid animals was reduced to the euthyroid level after T(3) treatment. The microtubular network of the mutant livers appeared, by immunofluorescence confocal microscopy, generally disorganized and drastically reduced in beta-tubulin in mice lacking TRbeta. In conclusion, our results indicate that beta-tubulin is critically controlled by TRbeta in the liver and that both TRs are probably needed to maintain the microtubular network organization of the liver.     

Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat lung cells is due to enhanced receptor synthesis

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung (FRL) cells is increased approximately 2 to 3-fold within 18 h of retinoic acid addition. Analysis of 125I-EGF binding assays at 0 C reveals approximately 25,000 receptors per cell, while analysis of growth factor binding to retinoic acid-treated cells demonstrates an increase in receptor levels to approximately 70,000 receptors per cell with no detectable changes in receptor affinities. We show by immunoprecipitation of 35S-methionine labeled EGF receptors that retinoic acid addition produces an increase in the accumulation of EGF receptor protein. Using brief pulses of 35S-methionine, an increase in EGF receptor synthesis can be identified within 3 h after retinoic acid addition. These results are the first to demonstrate that a retinoic acid-induced increase in 125I-EGF binding capacity is due to increased EGF receptor protein synthesis. Also, we find that a transient decrease in the rate of EGF receptor turnover occurs when retinoic acid is initially added to FRL cells. On the basis of our data, we conclude that the retinoic acid-induced accumulation of EGF receptors in FRL cells is primarily due to increased receptor synthesis. The effect of retinoic acid on EGF receptor turnover may be a secondary factor, influencing the rate at which receptors accumulate.     

Decreased expression of liver epidermal growth factor receptors in rats with alloxan and streptozotocin diabetes

Male rats (200 g) were rendered diabetic with one intraperitoneal injection of alloxan (150 mg/kg) or streptozotocin (60 mg/kg). In hyperglycemic animals within 3 hours after the injection, the binding of EGF to liver membranes decreased by 43-52%; the maximal drop was by 70% and persisted for the 20 days of the experiment. EGF receptors decreased in number with almost no changes in their affinity. Autophosphorylation of the receptors decreased parallel to the ligand binding. In animals that received lower doses and did not develop diabetes and in animals in whom diabetes was prevented by the injections of glucose (before alloxan) or nicotinamide (before streptozotocin) the binding of EGF to liver receptors remained normal. We conclude that the decreased expression of EGF receptors was caused by diabetes and not by the toxic effects of the diabetogenic compounds on the liver.     

Effect of streptozotocin-induced diabetes on epidermal growth factor receptors in rat liver plasma membrane

The effect of streptozotocin-induced diabetes on 125I-labeled epidermal growth factor (EGF) binding was studied in microsomal membranes from rat liver. The binding of EGF in membranes from diabetic animals was significantly low, the value being about 60% of the control level. Scatchard analysis of the binding data clearly showed that the decrease in EGF binding was due to a decrease in the number of receptors. Treatment of diabetic animals with insulin restored EGF receptors to control levels, whereas the treatment with triiodothyronine had no effect. Serum EGF concentrations measured were almost the same among the control, diabetic, and insulin-treated diabetic groups. These results suggest that insulin deficiency in vivo causes a decrease in hepatic EGF receptors.