Lynne Holtzclaw

Developmental regulation of insulin in the mammalian central nervous system

We delineated the ontogeny of rabbit brain insulin concentrations to define the regulatory role of development on this hormone in the central nervous system. Employing a sensitive ELISA, we observed higher concentrations in the late gestation fetal brain (approximately 80-90 ng/g) and early neonatal brain (approximately 195 ng/g) in comparison to the adult (approximately 32 ng/g; P less than 0.01). Further, we characterized this hormone to determine the identity of insulin (or an insulin-like substance) in brain. Employing porcine/bovine or rabbit insulin as standards, we observed that brain insulin mimicked authentic insulin in its migration on SDS-polyacrylamide and native gel electrophoresis, immunogenicity on Western blot analysis, and its elution profile on immunoaffinity column chromatographic, and high performance liquid chromatographic separation. We then examined the developmental effects on circulating and cerebrospinal fluid (CSF) radioimmunoassayable insulin levels. No statistically significant differences (ANOVA) existed through development in either the serum or CSF insulin levels. Employing multiple regression analysis, no correlation was evident between brain and either serum or CSF insulin concentration. A search for insulin mRNA by Northern blot analysis yielded minute amounts of atypical large sized transcripts. We conclude that the insulin peptide in the central nervous system closely resembles (or is identical to) circulating insulin in many properties and that there is a developmental increase in brain insulin concentrations, the maximal peak occuring in the late gestation fetus and early neonate. Insulin concentrations in brain demonstrate no conventional relationship to either the serum or CSF insulin levels, suggesting an additional source of peptide, which contributes beyond that which is available via the circulation. The amounts of insulin present within the central nervous system are minute (difficult to detect) but in the range (10-100 ng) where the hormone can interact with either insulin or insulin-like growth factor I (IGF-I) receptors that are abundantly present on developing brain cells, thereby executing the biological function of the hormone.

The neonatal rabbit brain glucose transporter

The Glut 1 (Hep G2/rat brain) isoform of glucose transporter is expressed in significant amounts in adult mammalian brain. The purpose of our present study was to determine the brain cellular localization of Glut 1 during the late newborn stage of development, when brain cellular proliferation and differentiation is highly active. Employing immunohistochemistry and in-situ hybridization in 10-day-old neonatal rabbit brain sections, we undertook cellular localization of Glut 1 expression. Glut 1 protein and mRNA were mainly noted in considerable amounts within the 10-day-old brain microvasculature. Lower concentrations of Glut 1 immunoreactivity were present in certain glial cells found within the deeper cortical layers of brain. Northern blot analysis of total RNA from isolated microvasculature-enriched preparation, isolated and cultured neuronal and glial cells, whole brain and whole brain with the exclusion of microvasculature obtained from the 10-day-old, revealed the universal presence of a approximately 2.8 kb Glut 1 mRNA with the exception of the neuron-enriched cultures. We conclude that during the neonatal period, when parenchymal cellular proliferation is at a peak, Glut 1 is localized not only to the microvasculature but also to certain cells which express glial morphological characteristics. The neuronal cells either do not express Glut 1 or express minute amounts.

The heterogeneity of the developing brain insulin receptor.

ABSTRACT: Comparison of the adult brain insulin receptor (IR) to other tissue IR demonstrates that the former migrates ∼10 kD faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to deficient sialic acid content of the asparagine N-linked carbohydrate moieties. We studied these receptors in the fetal rat (18-day) brain (∼125 kD) and liver (∼135 kD), and demonstrated that similar differences are present during fetal life. These differences are not modified by hyperglycemia associated with both mild hyperinsulinemia and normoinsulinemia/hypoinsulinemia. We further studied the specific brain cell types: neurons, glial cells, and purified microvessel preparation, and demonstrated a heterogeneity in the N-linked glycosylation of the IR within an organ (brain). The neuronal (∼125 kD) and microvascular (∼125 kD, ∼135 kD) IR are deficient in sialic acid, thus conferring neuraminidase-insensitivity to the whole brain, whereas the glial cell IR, similar to the liver IR, exhibits neuraminidase sensitivity and migrates intermediate (∼128 kD) to the liver and brain IR. The functional significance of this receptor heterogeneity between various tissues and cells within the same organ (brain) remains to be determined.

The developmental pattern of rabbit brain insulin and insulin-like growth factor receptor expression

To examine the effect of development on rabbit brain insulin and insulin-like growth factor (IGF) receptor expression, we characterized and quantitated receptor mRNAs by Northern blot analysis and affinity-labeled ligand bound receptors by SDS-PAGE and autoradiography. At various stages of development ranging from 23 to 30 day gestational (term approximately 31 days), 1 to 10 day postnatal ages and the adult, no change in the whole brain insulin receptor mRNA (7.0, 6.0 and 5.5 kb) and affinity-labeled receptor protein (approximately 125 kDa) levels was observed. The IGF-I receptor mRNA (11.5, 6.5 and 4.5 kb) and affinity-labeled receptor (approximately 125 kDa) protein levels declined during the neonatal stages of development. In the case of the IGF-II receptor, while the mRNA levels (9.0 and 4.5 kb) remained constant, the corresponding affinity-labeled receptor protein (approximately 230 kDa) declined with maturation. We conclude that a differential regulation of brain insulin, IGF-I and IGF-II receptor expression occurs during development.

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca2

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K+]o, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca2+]i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K+]o), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K+]o) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca2+]i induced by 55 mM [K+]o became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca2+]i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca2+]i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.