Oral Administration of a GSK3 Inhibitor Increases Brain Insulin-like Growth Factor I Levels

Reduced brain input of serum insulin-like growth factor I (IGF-I), a potent neurotrophic peptide, may be associated with neurodegenerative processes. Thus, analysis of the mechanisms involved in passage of blood-borne IGF-I into the brain may shed light onto pathological mechanisms in neurodegeneration and provide new drug targets. A site of entrance of serum IGF-I into the brain is the choroid plexus. The transport mechanism for IGF-I in this specialized epithelium involves the IGF-I receptor and the membrane multicargo transporter megalin/LRP2. We have now analyzed this process in greater detail and found that the IGF-I receptor interacts with the transmembrane region of megalin, whereas the perimembrane domain of megalin is required for IGF-I internalization. Furthermore, a GSK3 site within the Src homology 3 domain of the C-terminal region of megalin is a key regulator of IGF-I transport. Thus, inhibition of GSK3 markedly increased internalization of IGF-I, whereas mutation of this GSK3 site abrogated this increase. Notably, oral administration of a GSK3 inhibitor to adult wild-type mice or to amyloid precursor protein/presenilin 1 mice modeling Alzheimer amyloidosis significantly increased brain IGF-I content. These results indicate that pharmacological modulation of IGF-I transport by megalin may be used to increase brain availability of serum IGF-I. Interestingly, GSK3 inhibitors such as those under development to treat Alzheimer disease may show therapeutic efficacy in part by increasing brain IGF-I levels, an effect already reported for other neuroprotective compounds.     

Localization of the insulin-like growth factor I receptor in the cerebellum and hypothalamus of adult rats: an electron microscopic study

Insulin-like growth factor I (IGF-I) is an important modulator of cell growth and plasticity in the CNS. Expression of the IGF-I receptor mRNA in brain peaks at times of active cell development perinatally and remains detectable, albeit at lower levels, in the adult. While both autoradiographic and in situ hybridization studies show a wide and specific distribution of IGF-I receptor throughout the adult rat brain, nothing is yet known about its subcellular localization, a critical issue that will help clarify the biological role of this trophic factor in the adult brain. The present study describes the subcellular localization of IGF-I receptor immunoreactivity in the cerebellar cortex and the hypothalamic arcuate nucleus by using electron microscopic immunocytochemistry. In the cerebellum, IGF-I receptor immunoreactivity is present postsynaptically in the dendrites and soma of the Purkinje cell and presynaptically in axon terminals impinging upon the Purkinje cell soma, as well as in mossy fibre rosettes in the cerebellar glomeruli. Neurons in the mediobasal hypothalamus also contain IGF-I receptors located pre- and postsynaptically. Endothelial cells, astroglial end-feet surrounding micro vessels throughout all the brain parenchyma, tanycytes of the third ventricle and oligodendrocytes in the cerebellar white matter are also rich in IGF-I receptors. These results strongly support previous observations that IGF-I is a neuromodulator in the adult brain, probably acting as both a pre- and a postsynaptic messenger. They also suggest that glial cells may be involved in the actions of IGF-I in the adult brain.     

Effect of [D-Trp6]LHRH infusion on prolactin secretion by perifused rat pituitary cells

The effect of a superactive agonistic analog of luteinizing hormone-releasing hormone (LHRH), [D-Trp6]LHRH on prolactin (PRL) secretion by perifused rat pituitary cells was investigated. Constant infusion of [D-Trp6]LHRH (0.5 ng/min) for 2-3 h elicited a significant decrease in PRL secretion by these cells. This decrease in PRL release started ca. 30 min after the beginning of the infusion with the LHRH analog and lasted up to 1.5-2 h. [D-Trp6]LHRH significantly stimulated luteinizing hormone (LH) secretion during the first 30 min of peptide infusion; thereafter, LH levels began to return to control values. In animals pretreated in vivo with 50 micrograms of [D-Trp6]LHRH (s.c.) 1 h before sacrifice, PRL secretion by the rat pituitary cell perifusion system was significantly lower than vehicle-injected controls throughout the entire [D-Trp6]LHRH infusion period. On the other hand, thyrotropin-releasing hormone (TRH)-stimulated PRL secretion was slightly, but significantly imparied by [D-Trp6]LHRH infusion, while dopamine (DA) inhibition of PRL release was unaffected by this same treatment. These results reinforce previous observations of a modulatory effect of [D-Trp6]LHRH, probably mediated by pituitary gonadotrophs, on PRL secretion by the anterior pituitary. In addition, our findings suggest that basal PRL secretion by the lactotroph may be dependent on a normal function of the gonadotroph. The collected data from this and previous reports support the existence of a functional link between gonadotrophs and lactotrophs in the rat pituitary gland.     

Synthesis and biological properties of ovine corticotropin-releasing factor (CRF)

The 41-residue sequence of recently identified ovine corticotropin-releasing factor (CRF) was assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptide was purified by gel filtration and reverse-phase, medium pressure, followed by high-performance liquid chromatography (HPLC). In addition to the usual criteria, the homogeneity of the final material, obtained in 7% yield, was assessed by the isolation and examination of cyanogen bromide cleavage and tryptic digestion fragments by HPLC and amino acid analysis. The synthetic 41 amino acid CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column, the responses being related to the log-dose of CRF in the range of 0.05-125 ng/ml. The synthetic peptide also augmented in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal, as assessed by the measurement of serum corticosterone. The data indicates chemical purity and high biological activity of synthetic material.     

Synthesis and biological activity of a cyclic pseudohexapeptide analog of somatostatin

A cyclic pseudohexapeptide analog of somatostatin, cyclo(Pro psi[ CH2S ]Phe-D-Trp-Lys-Thr-Phe) was synthesized by solid phase methods and diphenylphosphoryl azide ring closure. The resulting crystalline compound possessed 23% of the growth hormone inhibitory activity of the parent tetradecapeptide and approximately 6% of the activity of the all-amide cyclic hexapeptide analog in spite of the absence of one of two postulated intramolecular hydrogen bonds.     

Stimulation of insulin secretion by corticotropin-releasing factor (CRF) in anesthetized rats

Corticotropin-releasing factor-containing cells have been recently found in the endocrine pancreas of several vertebrate species by immunocytochemistry. In order to clarify the possible physiological significance of these findings, we have studied the effect of the administration of CRF on endocrine pancreatic function. Five minutes, after injection of ovine CRF 1-41 into the jugular vein, a dose-related increase in insulin levels in the hepatic-portal vein of anesthetized rats was found. This dose-dependent insulin increase was delayed to fifteen minutes after CRF injection into rats exposed to greater surgical stress and was partially blunted in adrenalectomized animals. Glucose and glucagon levels were not altered after CRF administration under these conditions. These results suggest that CRF may play a modulatory role in insulin secretion; however, whether CRF acts directly on the beta-cell or through some CRF-stimulated mediator remains to be established.     

Serum insulin-like growth factor I regulates brain amyloid-beta levels

Levels of insulin-like growth factor I (IGF-I), a neuroprotective hormone, decrease in serum during aging, whereas amyloid-beta (Abeta), which is involved in the pathogenesis of Alzheimer disease, accumulates in the brain. High brain Abeta levels are found at an early age in mutant mice with low circulating IGF-I, and Abeta burden can be reduced in aging rats by increasing serum IGF-I. This opposing relationship between serum IGF-I and brain Abeta levels reflects the ability of IGF-I to induce clearance of brain Abeta, probably by enhancing transport of Abeta carrier proteins such as albumin and transthyretin into the brain. This effect is antagonized by tumor necrosis factor-alpha, a pro-inflammatory cytokine putatively involved in dementia and aging. Because IGF-I treatment of mice overexpressing mutant amyloid markedly reduces their brain Abeta burden, we consider that circulating IGF-I is a physiological regulator of brain amyloid levels with therapeutic potential.     

Insulin-like growth factors as mediators of functional plasticity in the adult brain.

Insulin-like growth factors (IGFs) are present in the brain throughout life. While their role as modulators of brain growth and differentiation during development is becoming apparent, their possible involvement in adult brain function is less known. Nevertheless, accumulating evidence indicates a role for IGFs in brain plasticity processes. Specifically, IGFs modulate synaptic efficacy by regulating synapse formation, neurotransmitter release and neuronal excitability. IGFs also provide constant trophic support to target cells in the brain and in this way maintain appropriate neuronal function. Pathological dearrangement of this trophic input may lead to brain disease. Molecular targets of the IGFs in the adult brain may include pre- and post-synaptic proteins involved in synaptic contacts, membrane channels, neurite-guiding molecules, extracellular matrix components and glial-derived intercellular messengers. Future studies on the role of IGFs in the adult brain may help unravel the relationship between neuronal plasticity and brain disease.     

Corticotropin Releasing Factor (CRF): Immunocytochemical localization and Radioimmunoassay (RIA)

Recent isolation, structural identification, and synthesis of ovine CRF has made possible the generation of specific antibodies against this hypothalamic peptide. Two fragments of the amino acid sequence corresponding to ovine CRF (CRF 37-41 and CRF 22-41), as well as the full sequence of 41 residues (CRF 1-41), synthesized in our laboratories by solid-phase methods, were coupled to bovine serum albumin (BSA) with glutaraldehyde. New Zealand white rabbits were immunized with the emulsified mixtures of peptide-BSA conjugates and Freund's adjuvant as immunogens. The specificity of the generated antibodies was studied by agar-gel diffusion, absorption tests in the immunohistochemical system, and with the aid of displacement curves in RIA. ¹²⁵I-Tyr(35)-CRF 36-41 and ¹²⁵I-Tyr(0)-CRF 1-41 were used as radioligands in the RIA. The minimum detectable dose was 20 pg. The linearity observed in RIA for immunoreactive CRF in extracts of rat hypothalami, together with the immunocytochemical findings in the rat brain, indicate the presence of substance(s) immunologically indistinguishable from CRF. Immunohistochemistry with the peroxidase-antiperoxidase (PAP) technique detected the following CRF-immunoreactive structures in vibratome sections of hypothalami of colchicine-treated rats: CRF-containing cell bodies were observed mainly in smaller neurons of the paraventricular nucleus. CRF-positive nerve fibers and/or terminals were present in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The CRF-positive terminals were localized in the central regions of the median eminence. These morphological data reinforce the view that this polypeptide plays a physiological role in the control of ACTH release.     

Radioimmunoassay of CRF-like material in rat plasma: validation of the method

The availability of antibodies against the ovine corticotropin releasing factor (CRF), which cross-react with a CRF-like immunoreactivity (CRF-LI) in the rat, has enabled us to develop a radioimmunoassay (RIA) for rat CRF-LI in plasma and crude hypothalamic extracts. 125I-Tyr CRF 1-41 was used as the tracer, and synthetic ovine CRF as the reference hormone. The precision profile of the assay indicates a high degree of reproducibility except for the lower dose range. The minimum detectable dose was 20 pg/tube. This assay can detect differences in plasma CRF-LI levels after various manipulations that simultaneously alter the ACTH levels in plasma. A wide range of CRF concentrations has been found in plasma of normal rats. Caution should be exercised in the interpretation of the values obtained since an ovine RIA system was used.