Immunohistochemical localization of insulin receptors and phosphotyrosine in the brainstem of the adult rat.

Previous studies have demonstrated that insulin receptors are widely distributed throughout areas of the forebrain in the adult rat that are involved in modulating neuroendocrine functions and feeding behaviour. In addition, a recent investigation showed that there is a good correlation between the presence of the insulin receptor and phosphotyrosine-containing proteins in these regions, indicating a possible functional activity of insulin receptors in vivo. It is unknown whether neural connections between specific brainstem nuclei to forebrain regions may also be under direct regulation of insulin or related factors. In order to test this possibility, the distribution of insulin receptors and phosphotyrosine was mapped throughout the hindbrain of the adult rat by immunocytochemistry, using specific antibodies against the beta-subunit of the insulin receptor as well as against phosphotyrosine. Both markers showed a high degree of overlap throughout numerous distinct anatomical regions of the hindbrain. In the mesencephalon, insulin receptor and phosphotyrosine-positive neurons were found in the precommissural nucleus, the lateral and dorsal part of the central gray, the mammillary bodies and the interpeduncular nucleus. In addition, immunoreactivity was found in the subependymal layer around the aqueduct along fibres and nerve cells possibly contacting the cerebrospinal fluid. In the pons and medulla, dense immunoreactivity was seen in the lateral superior olive, nucleus of the solitary tract, spinal trigeminal nucleus and nucleus ambiguous. Scattered cells were found in the pontine and vestibular nuclei, as well as in the reticular formation. The cerebellum contained moderately dense immunoreactivity in the granule cell and molecular cell layer of the cortex, as well as in the deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct beta-subunits are present in hybrid insulin-like-growth-factor-1 receptors in the central nervous system.

Previous work suggests the existence of different isoforms of the insulin-like-growth-factor-1 (IGF-1) receptor in various tissues. In the present study we provide support for the concept that heterogeneous IGF-1 receptors exist in the brain and that part of the heterogeneity is derived from IGF-1 receptor hybrids formed from different beta-subunits. IGF-1 receptors were extracted from adult-rat forebrain synaptosomes and partially purified by wheat-germ agglutinin (WGA) chromatography. Hormone-binding studies in this preparation demonstrate the presence of receptors for IGF-1 and insulin. An antibody, a-RIR, specific for the rat insulin receptor was used to remove insulin receptors from the WGA extract. Studies with the immunodepleted material demonstrated two proteins of 92 and 99 kDa that are phosphorylated on tyrosine during incubation with low concentrations of IGF-1. Both proteins bound with high affinity and specificity to IGF-1 immobilized on agarose, and each underwent phosphorylation when the agarose beads were incubated with [gamma-32P]ATP and MnCl2. Two-dimensional phosphopeptide maps after exhaustive trypsin treatment of the two proteins showed significant differences in their structure as well as differences from the phosphopeptide map for the beta-subunit of the insulin receptor. The relationship of the two proteins to the IGF-1 receptor was further probed by an antibody (a-HF) raised against a specific sequence in the beta-subunit of the human IGF-1 receptor, and a polyclonal antibody raised against the liver insulin receptor (L1) which cross-reacts with the IGF-1 receptor. Both antibodies immunoprecipitated the two phosphorylated proteins. However, reduction of the receptors to form receptor dimers or monomers showed that a-HF precipitated only the 99 kDa protein, whereas L1 precipitated primarily the 92 kDa protein. In conclusion, the brain IGF-1 receptor apparently has two structurally different beta-subunits, one of 92 kDa and a second of 99 kDa. Interestingly, at least a portion of the IGF-1 receptor population has both isoforms in the same receptor.

The hypophyseal pars tuberalis is enriched with distinct phosphotyrosine-containing proteins not detected in other areas of the brain and pituitary.

The regulation of cell activity, growth and metabolism by a number of growth factor receptors and proto-oncogene products involves tyrosine kinase activity resulting in autophosphorylation of the receptors and production of phosphorylated tyrosine-containing protein substrates. The identification and precise localization of phosphotyrosine (PY)-containing proteins are first steps in elucidating the functional role of tyrosine kinases in the modulation of the central nervous system and related areas. In the present report, we describe PY-containing proteins in the median eminence and adjacent pars tuberalis of the rat adenohypophysis by immunocytochemistry using light and electron microscopy, and by Western blotting analysis. PY-immunoreactivity was found to be most intense throughout the cytoplasm of a population of epithelial pars tuberalis cells. Polyacrylamide gel electrophoresis and Western blotting of tissue extracts from various brain and pituitary regions demonstrated a general pattern of 4 major bands of PY-proteins, with an additional dense band representing a 44 kDa protein that was highly phosphorylated on tyrosines and that was exclusively found in the pars tuberalis. Additional investigation for the presence of insulin receptors, a tyrosine kinase previously correlated with the distribution of PY-proteins, demonstrated a receptor localization in axons and nerve terminals in the external and internal zone of the median eminence. However, the large amount of different PY-proteins present in the secretory cell population of the pars tuberalis could not be attributed to the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Longitudinal gonadal steroid excretion in free-living male and female meerkats (Suricata suricatta).

Slender-tailed meerkats (Suricata suricatta) are small, diurnal, cooperatively breeding mongooses of the family Herpestidae. A prerequisite to fully understanding the mating system of meerkats is the development of a normative reproductive-endocrine database. This study examined longitudinal gonadal steroid excretion in all adult and juvenile individuals of both sexes within a social group of free-living meerkats sampled across an entire breeding season. The specific objectives of this study were to (1) validate noninvasive (fecal and urinary) gonadal steroid hormone monitoring techniques in male (testosterone) and female (estrogens, progestagens) meerkats; (2) test the feasibility of using these noninvasive methods under field conditions; (3) characterize the endocrine correlates associated with the female reproductive cycle, including estrus, gestation, and postpartum estrus; (4) examine longitudinal androgen excretion in males; and (5) determine whether social status (i.e., dominant versus subordinate) affected gonadal steroid excretion. In females, the results demonstrated the physiological validity of noninvasive monitoring in meerkats by corresponding excretory hormone concentrations to major reproductive events (i.e., estrous, pregnancy, parturition). Hormone excretory patterns during estrous intervals suggested possible mechanisms whereby reproductive suppression may operate in female meerkats. In males, androgen excretion did not correspond to changes in reproductive and aggressive behaviors, suggesting that dominance, and hence breeding access to females, was not regulated strictly by gonadal steroid production. The consistency in androgen excretion among male meerkats indicated that reproductive suppression may be mediated by behavioral (i.e., intermale aggression) rather than physiological (i.e., depressed spermatogenesis) mechanisms.

Location of phosphotyrosine-containing proteins by immunocytochemistry in the rat forebrain corresponds to the distribution of the insulin receptor.

Cellular regulation by certain growth factor receptors and protooncogene products involves tyrosine kinase activity with the resultant tyrosine phosphorylation of protein substrates. In the present report we describe the distribution of phosphotyrosine-containing material detected by immunocytochemistry (ICC) in the rat forebrain. Specificity of the affinity-purified antibody against phosphotyrosine used in the ICC technique was demonstrated by the ability of phosphotyrosine and p-nitrophenyl phosphate but not phosphoserine, phosphothreonine, or L-tyrosine to inhibit the immunostaining reaction. With ICC, relatively high amounts of phosphotyrosine-positive material were observed in neurons in specific structures that included the supraoptic, paraventricular, and arcuate nuclei; the median eminence; medial habenula; subfornical organ; and piriform cortex. Moderate to high amounts were present in the cerebral cortical layers II-IV and in the pyramidal cell layer of the hippocampus. Small to moderate amounts were detected in a few other locations. Glial elements showed minimal staining. Other areas of the rat forebrain failed to react with this antibody. Importantly, the distribution of the areas positive for phosphotyrosine agreed to a remarkable extent with the distribution of the brain insulin receptor, which itself has tyrosine kinase activity. These findings suggest a relationship between the insulin receptor and the increased phosphotyrosine content of these neurons and support the concept that the brain insulin receptor is active in vivo.