Genetically engineered mice in drug development.

Complex metabolic diseases like type 2 diabetes (noninsulin-dependent diabetes) and obesity present a difficult challenge to pharmaceutical companies in their attempts to develop new therapies. The polygenic nature of these diseases and the influence of nongenetic factors make it difficult to identify the most critical molecular processes to target for drug development. Transgenic animal models provide an approach to evaluate specific sites in metabolic and hormone signalling pathways under physiologic (as opposed to in vitro) conditions. The advantages and limitations of using transgenic animals in drug development will be covered and an overview of recent information from transgenic studies relevant to type 2 diabetes and obesity will be given.

Regional difference in insulin inhibition of non-esterified fatty acid release from human adipocytes: relation to insulin receptor phosphorylation and intracellular signalling through the insulin receptor substrate-1 pathway.

Increased mobilization of non-esterified fatty acids (NEFA) from visceral as opposed to peripheral fat depots can lead to metabolic disturbances because of the direct portal link between visceral fat and the liver. Compared with peripheral fat, visceral fat shows a decreased response to insulin. The mechanisms behind these site variations were investigated by comparing insulin action on NEFA metabolism with insulin receptor signal transduction through the insulin receptor substrate-1 (IRS-1) pathway in omental (visceral) and subcutaneous human fat obtained during elective surgery. Insulin inhibited lipolysis and stimulated NEFA re-esterification. This was counteracted by wortmannin, an inhibitor of phosphaditylinositol (PI) 3-kinase. The effects of insulin on antilipolysis and NEFA re-esterification were greatly reduced in omental fat cells. Insulin receptor binding capacity, mRNA and protein expression did not differ between the cell types. Insulin was four times more effective in stimulating tyrosine phosphorylation of the insulin receptor in subcutaneous fat cells (p < 0.001). Similarly, insulin was two to three times more effective in stimulating tyrosine phosphorylation of IRS-1 in subcutaneous fat cells (p < 0.01). This finding could be explained by finding that IRS-1 protein expression was reduced by 50 +/- 8% in omental fat cells (p < 0.01). In omental fat cells, maximum insulin-stimulated association of the p85 kDa subunit of PI 3-kinase to phosphotyrosine proteins and phosphotyrosine associated PI 3-kinase activity were both reduced by 50% (p < 0.05 or better). Thus, the ability of insulin to induce antilipolysis and stimulate NEFA re-esterification is reduced in visceral adipocytes. This reduction can be explained by reduced insulin receptor autophosphorylation and signal transduction through an IRS-1 associated PI 3-kinase pathway in visceral adipocytes.

Transgenic mice deficient in the LAR protein-tyrosine phosphatase exhibit profound defects in glucose homeostasis.

Protein-tyrosine phosphatases (PTPases) play an integral role in the regulation of cellular insulin action. LAR, a transmembrane PTPase expressed in insulin-sensitive tissues, acts as a negative regulator of insulin signaling in intact cell models. The physiological role of LAR was studied in mice in which LAR expression was eradicated by insertional mutagenesis. In the fasting state, adult male homozygous LAR (-/-) mice had significantly lower plasma levels of insulin and glucose, as well as a reduced rate of hepatic glucose production compared with wild-type controls, suggesting a heightened level of insulin sensitivity. In euglycemic clamp studies, the LAR (-/-) mice exhibited a significant resistance to insulin-stimulated glucose disposal and suppression of hepatic glucose output. Examination of hepatic insulin action demonstrated that the major alteration involved a 47% reduction in insulin-stimulated phosphatidylinositol 3'-kinase (PI 3-kinase) activity in the knockout mice, indicating a post-receptor signaling defect. Taken together with previous work on the cellular effects of LAR, the present results are consistent with a physiological role for LAR in the negative regulation of insulin action, with secondary abnormalities that contribute to the resistance to insulin-stimulated signaling in the knockout mice. Overall, these data provide further evidence for an important role for LAR in the regulation of insulin action and glucose homeostasis in intact animals.

Role of the glucagon receptor COOH-terminal domain in glucagon-mediated signaling and receptor internalization.

The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

Insulin signaling in mice expressing reduced levels of Syp.

Syp is a protein tyrosine phosphatase implicated in insulin and growth factor signaling. To evaluate the role of syp in insulin's regulation of plasma glucose, we generated knockout mice. Homozygous knockout mice die prior to day 10.5 of embryonic development. Hemizygous mice express half the levels of syp protein compared with their wild type littermates but do not display any gross morphological changes. Total body weight (age 2-10 weeks) and plasma insulin and glucose levels both in fasting and glucose-challenged states were comparable in the wild type and the hemizygous mice. No differences were observed in insulin-induced glucose uptake in soleus muscle and epididymal fat; insulin inhibition of lipolysis was also similar. We injected insulin into the portal vein of the mice to examine upstream events of the insulin signaling cascade. Tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) from hemizygous tissue was similar to that of wild type tissue. Association of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 increased an average of 2-fold in both groups. We did not observe an increase of IRS-1/syp association after insulin administration, but we did note a significant basal association in both wild type and hemizygous tissue. Our results do not support a major role for syp in the acute in vivo metabolic actions of insulin.

Differential effects of acute hypertriglyceridemia on insulin action and insulin receptor autophosphorylation.

Experimentally induced hypertriglyceridemia (HTG) and high plasma free fatty acid (FFA) levels impair in vivo insulin action. To determine if this is a consequence of impaired in vivo insulin receptor autophosphorylation and related to defective receptor signaling, hyperinsulinemic euglycemic clamps, indirect calorimetry, and skeletal muscle biopsies were performed in nine healthy subjects. In vivo insulin action was determined from the glucose infusion rate (GINF) and glucose oxidation (Glcox) during 40 and 120 mU/m2 /min clamps with (HTG clamp) and without (control clamp) a triglyceride emulsion infusion. The percentage of receptors autophosphorylated in vivo was determined by 125I-labeled insulin tracer binding in skeletal muscle immunoprecipitates of insulin receptors and phosphorylated receptors. Compared with the control clamps, plasma triglycerides and FFA increased four- and twofold, whereas GINF and Glcox decreased 15 and 35%, respectively, during the HTG clamps (all P<0.05). However, the percentages of receptors phosphorylated after the 40 and 120 mU/m2/min HTG clamps (9.2 +/- 1.5 and 21.1 +/- 2.6%, respectively) were similar to the control clamps (9.0 +/- 0.6 and 18.6 +/- 2.2%, respectively). These results indicate that, if impaired insulin signal transduction is a mechanism by which HTG and FFA impair insulin action, it occurs at a site downstream from insulin receptor autophosphorylation.

Glucagon.glucagon-like peptide I receptor chimeras reveal domains that determine specificity of glucagon binding.

The binding of glucagon to its hepatic receptor triggers a G-protein-mediated signal that ultimately leads to an increase in hepatic glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis). In order to elucidate the structural domain(s) of the human glucagon receptor (hGR) involved in the selective binding of glucagon, a series of chimeras was constructed in which various domains of the hGR were replaced by homologous regions from the receptor for the glucoincretin hormone, glucagon-like peptide I (GLP-IR). hGR and GLP-IR are quite similar (47% amino acid identify) yet have readily distinguishable ligand binding characteristics; glucagon binds to the recombinant hGR expressed in COS-7 cells with a Kd that is 1000-fold lower than the Kd for glucagon binding to GLP-IR. In the present study, chimeric receptors were transiently expressed in COS-7 cells and analyzed for glucagon binding. Expression of each receptor chimera was confirmed by immunofluorescence staining using a hGR-specific monoclonal antibody. This report identifies several non-contiguous domains of the hGR that are important for high affinity glucagon binding. Most notable are the membrane-proximal half of the amino-terminal extension, the first extracellular loop, and the third, fourth, and sixth transmembrane domains.

Deletion of lysine 121 creates a temperature-sensitive alteration in insulin binding by the insulin receptor.

Recently we reported the deletion of Lys-121 in one allele of the insulin receptor gene from a child with severe insulin resistance. In the present work, this mutant receptor (M121) was shown to have an abnormal sensitivity to temperature and an alteration in "negative cooperativity." In contrast to the wild-type receptor (HIRC), insulin binding by the M121 receptor was rapidly and irreversibly lost at temperatures above 30 degrees C with the phosphorylated form of the receptor being more temperature-sensitive than the nonphosphorylated form. Although insulin binding activity was lost, Western analysis and other studies showed that the mutant receptor remained intact. Measurements of 125I-insulin dissociation at 21 degrees C in the presence of native insulin (an estimate of negative cooperativity) demonstrated a difference between the mutant and wild-type receptor. Insulin dissociation from the mutant receptor was not as pronounced as that found with the wild-type receptor. Thus, an abnormality in insulin binding by the mutation was evident at lower "permissive" temperatures. The results of these and other studies argue that Lys-121 occupies an important position for the regulation of insulin receptor conformation. This regulation apparently influences negative cooperative interactions with insulin and modulates signal transduction.

Regulation of rat glucagon receptor expression.

This report describes the isolation of a cDNA for the rat glucagon receptor by using the glucagon-like peptide 1 receptor cDNA as a probe. Northern blot analysis using the cDNA clone showed that the message encoding the receptor is approximately 2.3 kb in size and is expressed only in liver and kidney among seven tissues tested. To study how glucagon receptor expression is regulated in vivo, the levels of hepatic glucagon receptor mRNA were measured in diabetic mouse model, db/db and control (db/+) mice. Interestingly, the receptor mRNA levels were similar between diabetic and control mice. In contrast, the number of hepatic glucagon receptors in diabetic mice measured by binding assays was significantly higher than that found in normal mice. These results suggest that the major regulation in hepatic glucagon receptor expression in vivo is at the posttranscriptional level.

Deletion of 3 basepairs resulting in the loss of lysine-121 in the insulin receptor alpha-subunit in a patient with leprechaunism: binding, phosphorylation, and biological activity.

We have identified a novel mutation of the human insulin receptor gene in a previously unreported patient with leprechaunism, leprechaun Rochester. This mutation consists of deletion of three nucleotides (GAA) in exon 2 and results in loss of the lysine-121 in the putative ligand-binding domain of the alpha-subunit. To analyze this mutation, we prepared a corresponding mutant insulin receptor by site-directed mutagenesis and expressed the receptor in Chinese hamster ovary cells. Although the mutant receptor displayed normal insulin binding, abnormalities were found in autophosphorylation and in phosphorylation of endogenous and exogenous protein substrates. These abnormalities consisted of increased basal kinase activity, but blunted insulin-stimulated responsiveness. Importantly, cells that expressed the mutant receptor showed markedly decreased insulin- and serum-stimulated DNA synthesis compared to untransfected control cells and cells transfected with the wild-type insulin receptor. These findings suggest that deletion of lysine-121 in conjunction with a presumed, but thus far unidentified, second mutant allele contributed significantly to the lethal insulin-resistant state in this patient with leprechaunism.

Association of the insulin receptor and phosphatidylinositol 3-kinase requires a third component.

We have studied the interactions between the insulin receptor and PtdIns 3-kinase by a reconstitution system in vitro composed of highly purified PtdIns 3-kinase from rat liver and highly purified insulin receptors bound to insulin-agarose or to antibodies against insulin receptors. As a positive control, receptors for platelet-derived growth factor, which bind and phosphorylate PtdIns 3-kinase, were studied in parallel with insulin receptors. Our results indicate that the insulin receptor, regardless of its phosphorylation state, does not directly associate with purified PtdIns 3-kinase, whereas the autophosphorylated receptor does associate with PtdIns 3-kinase present in the crude CHO-cell lysate. Also, we could not detect phosphorylation of PtdIns 3-kinase by the insulin receptor, even through the receptor readily underwent autophosphorylation and phosphorylated an insulin-receptor substrate, poly(Glu-Tyr) (4:1). These findings argue that one or more cytosolic components link the receptor and the enzyme. Insulin-receptor substrate-1 (IRS-1) was evaluated as a potential linking protein. In the absence of ATP, IRS-1 did not facilitate the coupling of the phosphorylated insulin receptor to PtdIns 3-kinase. Thus IRS-1 is unlikely to be the component in crude CHO-cell lysate that couples PtdIns 3-kinase to the phosphorylated insulin receptor. However, the addition of ATP, which allows phosphorylation of IRS-1 by the insulin receptor, also enhances the coupling of PtdIns 3-kinase to the insulin receptor. In support of this idea, immunoprecipitates of IRS-1 from insulin-treated CHO cells were found to contain both the insulin receptor and PtdIns 3-kinase. In conclusion, the insulin receptor does not appear to phosphorylate or bind directly to PtdIns 3-kinase, regardless of the receptor's state of phosphorylation. Association of PtdIns 3-kinase with the insulin receptor is mediated by one or more components, one of which may involve an unidentified factor in cell lysate and another that apparently involves phosphorylated IRS-1.

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)

Phosphotyrosine-containing proteins in the CNS of obese Zucker rats are decreased in the absence of changes in the insulin receptor.

The location and quantity of the insulin receptor and its associated tyrosine kinase activity have been examined in the forebrains of lean (Fa/?) and obese (fa/fa) Zucker rats using immunocytochemistry (ICC) and biochemical procedures. These studies were performed in conjunction with ICC and Western blot analysis of phosphotyrosine-containing proteins (PY-proteins). The results from ICC show a similar distribution and content for the insulin receptor among forebrain regions of lean and fatty Zucker rats. Biochemical analysis of the receptor was conducted on the hippocampus. Insulin binding studies using lectin-purified receptor extracts demonstrated similar receptor number and comparable hormone binding affinity for lean and obese animals. Autophosphorylation studies with the receptor extracts from the two groups did not find any differences in the tyrosine kinase activity of insulin receptors. In contrast to the normal findings with the insulin receptor, an abnormality in the obese animals was evident in the content of PY-proteins detected by ICC in the hippocampus, piriform cortex and olfactory bulb. Neurons in these brain regions showed a reduction in staining by an antibody against PY-proteins. Furthermore, Western blots of hippocampal extracts from obese rats demonstrated a reduction in phosphotyrosine content of two proteins of Mr 180 and 130 kD. These findings point to a previously unrecognized alteration in the CNS of the obese, insulin-resistant Zucker rat.

Mapping of carbohydrate sites on the human insulin receptor.

Prior to investigating the role of individual glycosylation sites in insulin receptor function, we are mapping the sites of glycosylation in the receptor. We report here a generally applicable methodology for the isolation and identification of glycosylation sites in cell surface glycoproteins. Human insulin receptors were labeled with [3H]-sugars using a CHO cell line transfected with the human receptor cDNA. Labelled receptors were mixed with receptors purified from human placental membranes and tryptic peptides prepared. Peptides were fractionated by gel filtration chromatography to limit the number of non-glycopeptides present. Peptides were then separated by reverse phase HPLC and glycopeptides identified by scintillation counting. Using this technique we have shown the insulin receptor to be glycosylated at Asn 397 and Asn 881. This increase the known number of occupied glycosylation sites to five.

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)

Acute effects of insulin-like growth factor I and insulin on glucose metabolism in vivo.

We have compared the actions of insulin-like growth factor (IGF-I) and insulin on glucose metabolism in vivo, using the glucose clamp technique in rats. Both hormones caused dose-dependent inhibition of hepatic glucose production, stimulation of whole body glucose disposal, and an increase in the glucose metabolic rate of specific muscles. Infusion of IGF-I also decreased the plasma concentration of insulin. An an infusion rate of 0.57 nmol.kg-1.min-1, IGF-I led to stimulation of whole body glucose uptake that was similar to the glucose uptake produced by infusion of 0.01 nmol.kg-1.min-1 insulin. The glucose metabolic rate, as measured by 2-deoxy-D-glucose uptake, was comparable in quadriceps femoris, soleus, and diaphragm muscles during the infusion of 0.57 nmol.kg-1.min-1 IGF-I and 0.01 nmol.kg-1.min-1 insulin. However, at these rates of infusion, IGF-I caused only a 38 +/- 6% inhibition of hepatic glucose output compared with 66 +/- 12% inhibition by insulin (P less than 0.05). Thus, under these conditions, muscle is more responsive than liver to IGF-I, which agrees with the complement of IGF-I receptors in the two tissues.

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.

Distribution of insulin receptor-like immunoreactivity in the rat forebrain.

Previous studies have suggested that insulin may play a role in the hormonal regulation of neurotransmitter metabolisms within the central nervous system. In order to provide additional information to support this hypothesis, we examined the distribution of insulin receptors within the forebrain of adult male rats. Insulin receptors were localized by immunocytochemistry, using an antibody directed against the carboxy-terminus of the beta-subunit of the insulin receptor. The antibody specificity was tested by immunoprecipitation of brain insulin receptors with antiserum and the purity of the receptor-antibody preparation was determined using hormone binding-assays with radiolabeled insulin and insulin-like growth factor-l. Insulin receptor-like immunoreactivity was found in a widespread, but selective, distribution on neurons throughout the rat forebrain. Double-labeling with glial fibrillary acidic protein did not demonstrate any detectable insulin receptor-like immunoreactivity on glial cells. Areas with the highest density of insulin receptor-like immunoreactivity were found in the olfactory bulbs, hypothalamus and median eminence, medial habenula, subthalamic nucleus, subfornical organ, CA 1/2 pyramidal cell layer of the hippocampus and piriform cortex. Double-staining of hypothalamic sections with somatostatin and vasopressin antisera revealed insulin receptor-like immunoreactivity on a subpopulation of somatostatin neurons in the periventricular region and on vasopressin neurons in the supraoptic nucleus. A moderately dense insulin receptor-like immunoreactivity was observed in layers II-IV of cerebral cortex, medial amygdala, reticular thalamic nucleus, zona incerta, and preoptic and septal regions, whereas a low density of insulin receptor-like immunoreactive neurons was found in basolateral amygdala and most thalamic regions. The basal ganglia and most parts of the thalamus were almost devoid of insulin receptor-like immunoreactivity. Our findings provide morphological support for a direct action of insulin on selected regions of the rat forebrain and suggest that the insulin receptor may modulate synaptic transmission or the release of neurotransmitters and peptide hormones in the CNS.