Brian A. Masters

Insulin receptors in the brain: structural and physiological characterization.

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of125I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t1/2 of dissociation of 26 minutes. Cross-linking of125I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the β subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [3H]norepinephrine uptake. Insulin inhibition of [3H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in theKm, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.

Insulin receptors and insulin action in dissociated brain cells

The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [125I]insulin to cells obtained by mechanically dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t1/2 of association was 14 min and t1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [125I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the alpha-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver alpha-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [3H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [3H]NE uptake was sodium ion-dependent suggesting that 80-90% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (Glu, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the beta-subunit of the insulin receptors. These observations corroborate our contention that insulin exerts neuromodulatory effects mediated by the specific insulin receptors in the brain.

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.

Expression of human metallothionein-III in transgenic mice

Transgenic mice that express human metallothionein-III (hMT-III) were generated. Human MT-III mRNA expression was prominent in brain, resulting in a 9-fold elevation of MT-III mRNA in cortex, a 3-5-fold elevation in hippocampus, thalamus, brainstem, and olfactory bulb, and a 1.4-fold elevation in cerebellum. Human MT-III protein was detected biochemically and accounted for a 3.4-fold increase in total brain MT. The concentration of zinc (but not copper) was elevated in those brain regions that expressed the most hMT-III mRNA. The histochemically reactive pool of zinc, as measured by Timms stain or TS-Q histofluorescence, was not appreciably altered. No changes in brain weight, morphology or histology have been noted; the mice breed normally and appear to have normal behavior.

Insulin-like Growth Factor I Receptors and IGF-I Actions in Neuronal Cultures from the Brain

Neurons in primary culture have been used to study IGF-I receptors and IGF-I-induced cellular actions in the brain. Intact neurons in culture specifically bind [125I]IGF with high affinity. The potency for the competition of [125I]IGF-I binding was IGF-I > IGF-II > insulin. A curvilinear Scatchard plot represented high-affinity (0.15 nM) and low-affinity (3 nM) binding sites with a Bmax of 142 fmol and 618 fmol/mg protein, respectively. These binding sites are predominantly localized on neurites with relatively few sites on the cell soma. IGF-I induced synthesis of protein(s) in the M(r) range of 48,000-50,000 with pI values of 6.1-6.4. These observations show that IGF-I receptor mediates induction of specific proteins and suggest that these proteins may be involved in the neurotrophic activity of IGF-I in the brain.

Insulin-Like Growth Factor Receptors in the Central Nervous System: Phosphorylation Events and Cellular Mediators of Biological Function

A complex pattern of growth and development in the nervous system would seem inherent in consideration of its morphological design for control of physiological processes and psychological phenomena. Yet in spite of this tangled design it would appear reasonable that certain basics tenets must be preserved in development and growth in the cellular world. Using such reasoning, the complex mechanisms responsible for growth and development in the nervous system have begun to acquire a very rudimentary definition. It has become increasingly apparent that a functional family of peptides termed growth factors are basic to the regulation of growth and development in peripheral cell populations (Daughaday, 1989). Recent research addressing nervous system growth and development points at these same peptide factors as important determinants in the generation and maintenance of the distinct cell types that comprise the brain, peripheral nerves, and organs of neural ectodermal origin.

Rat Brain/Hep G2 Glucose Transporter Gene Expression in Brain

Publisher Summary This chapter presents a methodology to quantitate Glut-1 mRNA and protein levels in the brain and in neuronal and astroglial cells in primary culture. Measurement of Glut-1 mRNA levels include isolation of total RNA, cDNA probe preparation, Northern blotting, and hybridization. In a study described in the chapter, a single ∼2.8-kb band that hybridized with Glut-1 cDNA probe was observed at each stage of brain development. The steady state levels of Glut-1 mRNA were relatively low at embryonic day 20 and at early postnatal stages. These levels increased around weaning time (postnatal day 21) and reached maximal values in the adult brain. Using scanning densitometry, it was found that Glut-1 mRNA levels in the adult brain were three- to fourfold higher than at birth. The chapter also discusses the measurement of Glut-1 protein by western blotting. It presents an autoradiogram of a representative experiment where Glut-1 protein levels have been measured in astroglial cells in response to the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA). TPA stimulation of [ 3 H]deoxy-D-glucose uptake in astrocytes is associated with an increase in the amount of Glut-1 protein.