Influence of nucleophosmin/B23 on DNA binding and transcriptional activity of the androgen receptor in prostate cancer cell.

The promotion and progression of prostate cancer (PCa) are associated with androgen receptor (AR) signalling. AR functions are modulated by a variety of co-factors amongst which we identified the nucleophosmin (NPM/B23), a member of the histone chaperone family. Here, we show that NPM is overexpressed in PCa compared to normal adjacent tissues. AR and NPM interact in vitro and in vivo, and NPM is critical for androgen-dependent transcriptional activation in LNCaP cells as an anti-NPM siRNA downregulates transcription of a transfected androgen response element (ARE)-containing reporter promoter as well as expression of the endogenous androgen responsive prostate-specific antigen (PSA) gene. By investigating the effect of NPM on AR, we have also observed that NPM enhances AR binding to an ARE in vitro in electrophoretic gel mobility-shift assay experiments. Chromatin immunoprecipitation studies further demonstrated that both AR and NPM associate with AREs of the PSA gene in vivo. Altogether, our data suggest that the molecular histone chaperone NPM could regulate AR functions by promoting assembly of AR-containing regulatory complexes and that high levels of NPM might alter AR functions in PCa.

Spontaneously immortalized epithelial cells from mouse caput epididymidis.

We report here on the characterization of tissue-culture cell lines derived from primary cultures of the mouse caput epididymidis epithelium. The cell lines were spontaneously immortalized without the use of transforming oncogenes. In defined conditions, our epididymal cells adopted various morphological features that resembles that of the in vivo epididymis epithelium such as a polarized organization and the presence of junctional structures at their apical/lateral membranes as revealed by electron microscopy analyses. Flow cytometry analysis revealed that we were dealing with homogenous cell populations that had reached a near-tetraploid state. RT-PCR assays were used in order to show that several genes that can be considered as markers of in vivo caput epididymidis epithelium activity were expressed in our cell lines confirming that these cells were indeed in a differentiated state close to their endogenous state.

Hormonal and developmental regulation of the mouse aldose reductase-like gene akr1b7 expression in Leydig cells.

The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0.25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.

Mouse seminal vesicle secretory protein of 99 amino acids (MSVSP99): characterization and hormonal and developmental regulation.

Polyclonal antibodies have been generated to investigate the localization, tissue and species distribution, androgen regulation, and ontogeny of a protein secreted by mouse seminal vesicle, designated as MSVSP99 (ie, mouse seminal vesicle secretory protein of 99 amino acids). MSVSP99 is a polymorphic compound with a molecular weight of around 14 kilodaltons and a positive immunoreactivity range of 5.23 to 5.70. Positive immunoreactivity was restricted to the epithelial cells of the seminal vesicle. Western blot analysis showed organ specificity for MSVSP99, which could not be detected in several organs in the mouse. Time course decrease of MSVSP99 after castration closely followed that of its mRNA. In contrast, the length of time required to restore control levels after testosterone treatment was higher for the protein than it was for its mRNA. Whereas the MSVSP99 gene is already active in 10-day-old males, MSVSP99 is first detected at 27 days. Then, we conclude that factors other than the accumulation of the mRNA regulate MSVSP99 expression.

Acquisition of androgen-mediated expression of mouse vas deferens protein (MVDP) gene in cultured epithelial cells and in vas deferens during postnatal development.

We used cultured vas deferens epithelial cells (VDECs) as a model system to determine the conditions that allow mouse vas deferens protein (MVDP) gene expression and acquisition of androgen responsiveness. On the basis of Northern blot analysis, the mvdp gene is constitutively expressed at very low levels in prepubertal VDECs grown on collagen-coated plastic or on microporous membrane inserts. In the presence of dihydrotestosterone (DHT), mvdp messenger RNA levels dramatically increased in cells cultured on microporous membrane inserts and stayed unchanged in cells grown on matrix-coated plastic. Epithelial cells derived from fetal vas deferens were able to synthesize MVDP in response to DHT, and the presence of fetal mesenchymal cells did not influence MVDP production. Providing the cells with a culture procedure that permits access to the basolateral membranes and caters to the polarity requirements of the cell is a prerequisite for androgen induction of MVDP gene expression. The results also point to a role for epidermal growth factor, insulin, and tyrosine kinase activity in mediating the action of androgen on mvdp gene expression. In vivo studies show that the first expression of the mvdp gene between 5 and 7 days postpartum is not associated with major structural changes in the epithelium. The acquisition of a mature phenotype by epithelial and peritubular contractile cells, between 10 and 20 days, correlates with androgen dependency of the mvdp gene. We propose that cell differentiation and polarization on a matrix-coated microporous membrane reproduces some of the events that are necessary for acquisition of androgenic responsiveness of the mvdp gene during postnatal development.

Down-regulation of AP1 activities after polarization of vas deferens epithelial cells correlates with androgen-induced gene expression.

Vas deferens epithelial cell subcultures were used to study the sequential regulation of jun/fos proto-oncogene expression and AP1 activities during cell proliferation, polarization and androgen-induced expression of a terminal differentiation marker, i. e. the mvdp gene. Proliferation of epithelial cells is associated with a high expression in the nucleus of most Jun and Fos oncoproteins. After cell seeding on an extracellular matrix which allows polarization and expression of the mvdp gene in response to androgens, AP1 protein accumulation is greatly altered and consists in a loss of JunB, Fra1, FosB and a decrease in c-Fos, c-Jun and Fra2, while JunD remained at the same level. This was correlated with a drop in AP1 binding activity as evaluated by gel shift assay using either AP1 consensus sequence or AP1 binding sites of the mvdp gene promoter region, and in AP1 transactivating activity, as estimated by stable transfection experiments using an AP1 responsive promoter (TRE-TK-luc). Androgens did not significantly influence AP1 activities. On the contrary, stimulation of AP1 proteins by the tumor-promoting phorbol ester caused a decrease in androgen-induced mvdp mRNA accumulation, and this effect was reversed by staurosporine, a potent inhibitor of PKC. Our data suggest that a down-regulation of AP1 activities induced by epithelial cell differentiation is a prerequisite to androgen-induced mvdp gene expression. The high AP1 activities observed during proliferative state or induced in TPA-treated polarized cells, exert a repressive effect on androgen action.

Morphological changes in the vas deferens and expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in control, deltaF508 and knock-out CFTR mice during postnatal life.

The morphology of the mouse vas deferens still undergoes major changes from birth to 40 days of age, such as differentiation of the mesenchymal cells into fibroblasts and muscle cells, differentiation of the epithelium into basal and columnar epithelial cells, development of stereocilia, and the appearance of smooth endoplasmic reticulum organised in fingerprint-like structures or parallel, flattened saccules. In mutant homozygous DeltaF508 (DeltaF/DeltaF) and knock-out (cf/cf) CFTR mice, strain 129/FvB and 129/C57BL-6, respectively, a similar development occurred until the age of 20 days. At 40 days, however, the lumen was filled with eosinophilic secretions, and sperm cells were absent in the majority of the animals examined, although sperm production in testis and epididymis appeared to be normal. CFTR was localised in the apical membrane and cytoplasm of the vas deferens epithelium from 40 days on but could not be detected in the vas deferens before 20 days or in mutant adult CFTR mice as expected. Western blots of membrane preparations showed that the mature form of CFTR was present in vas deferens and testis but absent in seminal vesicles. Our results suggest that the function of CFTR is probably essential after 20 days in the vas deferens and that its absence or dysfunction may result in a vas deferens with a differentiated epithelium but a collapsed lumen, which could at least temporarily delay the transport of spermatozoa. These observations contrast with those made in the overall majority of CF patients. Mol. Reprod. Dev. 55:125-135, 2000.

[Three therapeutic recipes from the pages of the baptismal record book of the Kastel Parish in Istria]. / Tri recepta sa stranica maticne knjige krstenih zupe Kastel u Istri.

In Croatian archives a rich collection of registers is preserved. Among the oldest and best-conserved collections of such valuable sources in Europe, are those from the territory of Istria. Investigating these sources we focused our attention on three recipes for treatment of calculi and cuts found on pages of Kastel baptismal's record (1749-1815) in Istria. Similar to other recipes found in various other recipe collections they mirror interlace of folk experience and theurgical views of healing which was detected unexpectedly sometimes on unconventional places, have survived on Croatian territory throughout centuries.

Androgens decrease and retinoids increase the expression of insulin-like growth factor-binding protein-3 in LNcaP prostatic adenocarcinoma cells.

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

5'-flanking and intragenic sequences confer androgenic and developmental regulation of mouse aldose reductase-like gene in vas deferens and adrenal in transgenic mice.

The MVDP (mouse vas deferens protein) gene, which encodes an aldose reductase-like enzyme, is mainly expressed in vas deferens epithelium and adrenal cortex. Vas deferens MVDP gene transcription was known to be under androgenic control, we now have evidence for androgen and probable ACTH responsiveness of the MVDP gene in the adrenal. To analyze the role of potential regulatory regions in hormonal, developmental, and tissue-specific aspects of MVDP regulation, we generated transgenic mice harboring MVDP-CAT fusion genes. The constructs carried either -1.8 or -0.5 kb 5'-flanking sequence attached to the chloramphenicol acetyltransferase gene in presence or absence of a 3.5-kb intragenic fragment in a downstream position. We show that at least two regions ensure proper gene regulation in vivo. The first, located within the 1.8-kb promoter fragment, directs tissue specificity; positive elements necessary for vas deferens and adrenal expression lay within positions -1804 to -510 and -510 to +41, respectively. The second, located within the 3.5-kb intragenic fragment spanning intron 1 to intron 2, increases percentage of expressing lines and behaves as a vas deferens-specific enhancer. Hormonal and developmental control of transgenes closely parallel endogenous gene regulation. Androgen and ACTH responsiveness in adrenals is conferred by 0.5-kb promoter, whereas in vas deferens, full androgenic response of the 1.8-kb promoter required the 3.5-kb intragenic fragment. Thus, vas deferens and adrenals use distinct cis-acting elements to direct and regulate the expression of the MVDP gene.

Ubiquitous transcription factors NF1 and Sp1 are involved in the androgen activation of the mouse vas deferens protein promoter.

Transcription of the mouse vas deferens protein (MVDP) gene is stimulated by androgens and we have previously shown that in a 162 bp fragment, located at position -121 to +41, a TGAAGTtccTGTTCT sequence functions as an androgen-dependent enhancer. To determine which factors are involved in the hormonally regulated MVDP gene transcription, we have used DNase I footprinting and band-shift assays to examine in vitro binding of proteins to the enhancer and promoter sequences and have determined the functional significance of the recognized DNA sequences in transient transfection assays. Studies using recombinant proteins such as the DNA binding domain of the androgen receptor (AR-DBD) and Sp1, and crude cellular extracts from T47D and vas deferens epithelial cells (VDEC) showed that in addition to AR-DBD, the transcriptional activators NF1 and Sp1 interact with the -121/+41 fragment in a specific manner. Transient transfection assays revealed that site-directed mutations in the NF1 and Sp1 binding elements strongly reduced (NF1) or abolished (Sp1) androgen induced expression. The results demonstrate that the -121/+41 sequence is a composite site for the androgen receptor mediated transactivation of the MVDP gene.

Vas deferens epithelial cells in subculture: a model to study androgen regulation of gene expression.

The understanding of androgen-regulated gene expression requires a cell cultures system that mimics the functions of cells in vivo. In the present paper we have examined a vas deferens epithelial cell subculture system. Cultured vas deferens epithelial cells have been shown to exhibit polarized properties characteristic of functioning epithelia and to display a high level of androgen receptors. Incubation of cells with androgen caused a decrease in cellular androgen receptor mRNA that was time-dependent. Total suppression was observed after 24h of exposure to androgen. By contrast, incubation of vas deferens epithelial cells with androgen resulted in a threefold increase in the cellular content of androgen receptor protein, as assayed by ligand binding. In response to androgens, vas deferens epithelial cells expressed mouse vas deferens protein mRNA (MVDP mRNA). Maximum expression of the MVDP gene, at both mRNA and protein levels, was observed after 24h of androgen induction. DEAE-dextran transfection conditions were defined using the MMTV-CAT gene in vas deferens epithelial cells in a dose- and time-dependent manner. No induction was seen when fragments of the MVDP promoter region were cloned directly in front of the CAT gene and transiently transfected into vas deferens epithelial cells. It was found that cotransfection of cells with MVDP-CAT gene and transfection of cells with MVDP-CAT constructs and with an androgen receptor expression vector resulted in a small but consistent androgen-dependent increase in reporter gene activity. Transiently transfected vas deferens epithelial cells are a suitable model with which to study the effect of androgen on gene regulatory elements.

Exportation of mouse vas deferens protein, a protein without a signal peptide, from mouse vas deferens epithelium: a model of apocrine secretion.

Mouse vas deferens protein (MVDP) is a major androgen-dependent protein of deferential fluid. It is specifically expressed in the epithelium of the mouse vas deferens. Its amino acid sequence as deduced from the nucleotidic sequence of its cDNA does not possess a signal sequence characteristic of secretory proteins. In vitro, transcription of MVDP cDNA followed by translation of mRNA in the rabbit reticulocyte system, in the absence or the presence of microsomes, demonstrated that there was no internalization of MVDP into microsomes that could protect it from degradation by proteinase K; this confirmed the absence of signal sequence. Moreover, MVDP has its NH2-terminus blocked. To understand how MVDP can be exported, its ultrastructural distribution and secretion process were analyzed by means of electron microscopy. Immunolocalization of MVDP revealed that it was distributed in the whole cytoplasm; it was never detected in the lumen of endoplasmic reticulum, Golgi apparatus, or vesicles but was abundant in apical protrusions and in the fluid, where it was associated with cellular material undergoing degradation. These data clearly demonstrated that exportation of MVDP into the luminal fluid does not occur in the classical manner for secretory proteins but rather involves an apocrine secretion process.

Identification of a functional androgen response element in the promoter of the gene for the androgen-regulated aldose reductase-like protein specific to the mouse vas deferens.

Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the epithelial cells of the deferent duct under androgenic regulation. To better understand androgenregulated MVDP gene expression, the location and sequences of androgen response elements (AREs) in the 5'-flanking DNA were determined. Sequence analysis revealed two putative AREs as follows: one between positions -1186 and -1171 (distal ARE) and the other between -111 and -97 (proximal ARE). To study hormonal regulation, fragments of the MVDP promoter region, extending from residue -1804 to +41, were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a human androgen receptor expression vector into T47D cells in a transient expression assay. A minimal region (-121 to +41) was identified as being sufficient for androgen-regulated gene expression. A mutation in proximal ARE almost completely abolished androgen induction of CAT. One copy of the sequence TGAAGT tcc TGTTCT, cloned in the opposite orientation in front of the thymidine kinase promoter, confers androgen responsiveness to the CAT reporter gene. Androgen transcriptional activity was not detected with the distal ARE. The data provide strong evidence that transcriptional regulation of the MVDP gene occurs via the sequence TGAAGT tcc TGTTCT.

Androgens regulate expression of the gene coding for a mouse vas deferens protein related to the aldo-keto reductase superfamily in epithelial cell subcultures.

Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the vas deferens. To better understand androgen-regulated MVDP gene expression we have used RNA hybridization to study the effects of androgens on the steady-state levels of MVDP mRNA in vas deferens epithelial cell subcultures. Northern blot analysis revealed that these cells only express MVDP mRNA in the presence of androgens. There was a close relationship between MVDP mRNA levels and dihydrotestosterone concentrations. MVDP mRNA is induced over a period of 24h and maximal induction is about 25-fold. Treatment of cells with cycloheximide completely abolished the observed androgen effect suggesting that the induction of the MVDP gene by androgens depends on continuous protein synthesis. Transient transfection of vas deferens epithelial cells with MMTV-CAT vector showed that these cells contained functional androgen receptors and that they are a suitable system to study androgen effect on MVDP gene regulatory elements.

In vitro androgenic induction of a major protein in epithelial cell subcultures from mouse vas deferens.

Pure epithelial cell cultures, obtained from primary culture of vas deferens tissue collected from 20- to 30-day-old mice, were amplified by subculturing the cells over 3T3 feeder layer in a serum-free defined medium. Adhesion and proliferation of epithelial cells did not require androgens, but a minimal concentration of 5.10(-7) M hydrocortisone. In that system, epithelial cells expressed cytokeratin but failed to produce the tissue specific mouse vas deferens protein (MVDP) in response to androgens. Various culture procedures and medium compositions were assayed for induction of MVDP expression. Culture onto microporous membrane inserts, which allow polarization of cells, is absolutely required for androgenic induction of MVDP. Androgen action did not require the presence of hydrocortisone, insulin, triiodothyronine, pituitary extracts, epidermal growth factor and acetylcholine. A minimal supplemented medium was then defined in which the expression of MVDP by epithelial cells in response to androgens was dose dependent. It has also been shown that this response at each concentration of dihydrotestosterone was heterogeneous at individual cell level. Highly reproducible results were obtained from epithelial cell cultures between 8th to 16th passages, showing that subcultured cells have maintained their ability to differentiate and express specialized functions.

Developmental and hormonal regulation of specific proteins in mouse vas deferens and seminal vesicle.

This paper is concerned with hormonal regulation of the developmental pattern of major proteins of the mouse vas deferens (mouse vas deferens protein: MVDP, 34.5 kD) and seminal vesicle (15.5, 120 and 140 kD) whose expression is regulated by testosterone at adulthood. The ontogeny of these proteins, studied by SDS-polyacrylamide gel electrophoresis, appeared to be uncoordinated. MVDP was not accumulated until animals were 20 days old and its concentration increased sharply from 20 to 30 days of age. In seminal vesicle, the 15.5 kD protein did not accumulate before day 30 whereas 120 and 140 kD proteins appeared and accumulated between 30 and 40 days. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP levels were not abolished and were similar to those measured in 20-day-old males. Testosterone administration, from 1 to 10 days of age, did not induce precocious expression of MVDP. These results suggest that the neonatal expression of MVDP is independent of androgens. In seminal vesicle, the first expression of the 3 proteins studied was dependent upon testicular androgens as shown by neonatal castration and injection experiments. The marked increase in the levels of the 4 proteins studied, during sexual maturation, was not associated with quantitative or qualitative changes in tissular androgen concentrations, suggesting that other factors may be necessary for protein expression. Whereas thyroxine may induce a precocious accumulation of MVDP, prolactin had no stimulatory effect on the accumulation of proteins from vas deferens and seminal vesicle. The results suggest that during sexual maturation gene activation by androgens was progressive.

Influence of low- and high-protein diets on insulin and insulin-like growth factor-1 binding to skeletal muscle and liver in the growing rat.

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.

Effect of calorie restriction on skeletal muscle and liver insulin binding in growing rat.

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 receptor tyrosine kinase activity in rat liver and skeletal muscle: effect of starvation.

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.