Ruben Schechter

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin I mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNAs within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.

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.

Insulin and insulin mRNA are detected in neuronal cell cultures maintained in an insulin-free/serum-free medium.

We investigated the effect of a serum-containing medium, exogenous insulin, and insulin-free/serum-free media in the regulation of the rabbit brain insulin-like peptide (ILP) in neuronal cell cultures. The presence of serum or insulin in the medium resulted in approximately 3-5% of neurons that were positive for the peptide by immunohistochemistry and for insulin mRNA by in situ hybridization. The absence of insulin in the medium resulted in a three- to fourfold increase (p less than 0.001) in the insulin-immunoreactive and insulin mRNA-containing neurons. Additionally, in the presence of exogenous insulin or serum, the amount of insulin present in the medium, as measured by ELISA, decreased with time (approximately 80%), the former slower than the latter when compared with their respective zero time point values. However, an increase (approximately 80% from zero time) was noted in the absence of insulin or serum, and this lasted for 24-48 hours alone. The presence of an increased insulin/ILP content in the medium and the increase in the numbers of insulin-immunoreactive neurons suggests an important role of the peptide in the brain. The observation of increased synthesis and secretion of neuronal ILP in the absence of insulin is indicative of an autocrine effect of exogenous insulin on neuronal production of ILP, which in turn may be important in the growth and maintenance of neuronal and possibly glial cells.

Neuronal synthesized insulin roles on neural differentiation within fetal rat neuron cell cultures.

We previously, described the production and secretion of insulin by fetal neurons in culture and demonstrated that neuronal synthesized insulin [I(n)] promoted neurofilament distribution and axonal growth. In this study we investigated the role of I(n) in promoting neural differentiation. Stem cells from 16 day gestational age rat brains were cultured in an insulin-free defined medium (IFDM) and treated with: 5, 20 or 100 ng/ml of exogenous insulin, 100 ng/ml insulin-like growth factor I (IGF-I) or an anti-insulin antibody. The neurons were studied at 1 and 3 days of incubation. The total number of cells showed no significant difference (P>0.05) in any of the media used, except the IFDM at day 3 of incubation treated with the anti-insulin antibody (P<0.05) and IGF-I to 20 ng/ml of insulin (P<0.05). No significant difference (P>0.05) was found in the number of differentiated neurons incubated in the IFMD, in which the neurons produce and secrete I(n), between days 1 and 3 of incubation, but neural differentiation decreased significantly (P<0.05) when treated with the anti-insulin antibody. Exogenous insulin significantly increased (P<0.05) the number of differentiated neurons compared to the IFDM. A significant reduction (P<0.05) of differentiated neurons was observed at day 3 of incubation with IGF-I compared to all the different media. Thus, I(n) has a role in promoting neural differentiation and growth, but exogenous insulin promoted neural differentiation and growth beyond I(n).

Insulin effects on extracellular signal regulated kinase cascade in fetal rat astrocyte cell cultures.

Insulin within the nervous system promotes cell differentiation and survival. In addition extracellular signal regulated kinase (ERK) promotes cell differentiation and survival. We studied the effects of insulin on astrocyte ERK-1 and 2, in fetal enriched astrocyte cell cultures incubated in an insulin free-defined medium (G3 medium). After 2 days in G3 medium the astrocytes were stimulated with 5 ng/ml of insulin for 2 min, and insulin effects on insulin receptor, insulin receptor substrate-1 (IRS-1) and ERK-1 and 2 were studied using Western blots. Insulin inhibited the phosphorylation of the insulin receptor and IRS-1 and decreased the activity of the ERK-1, and inhibited the activation of the ERK-2. Thus, insulin is involved in ERK regulation in fetal astrocytes.

Brain Endogenous Insulin Phosphorylated MAPK and Affected Axonal Growth in Fetal Rat Neuron Cell Cultures † 480

Brain Endogenous Insulin Phosphorylated MAPK and Affected Axonal Growth in Fetal Rat Neuron Cell Cultures † 480

ANTIBODY STAINING AND IN SITU HYBRIDIZATION REVEALS THAT A SUBSET OF NEURONS IN THE RABBIT NEONATAL BRAIN PRODUCE INSULIN

We have previously shown that insulin is present in whole brain extracts of fetal/neonatal rabbits (BBRC 136:208, 1986). However, the source of this hormone remains controversial. To resolve this issue, glia and neurons from 10 day old newborn rabbits were grown in tissue culture and subsequently analyzed for hormone synthesis by the peroxidase-antiperoxidase (PAP) technique and in situ HYB. Glia and neurons were identified on the basis of morphology and positive staining with enolase (neurons) and fibrillary acidic protein (glia). The monoclonal antibody (1:100) used in the PAP reaction was specific for insulin. In situ HYB was conducted at 37°C under stringent conditions using a biotin-labeled rat insulin cDNA probe (Dr. A. Permutt). Biotinylated pBR322 DNA served as a control. HYB was detected using avidin-Peroxidase. The data show that insulin is present in only ∼5% of the neurons but not glia. Pretreating the cells with the ionophore monensin, to block hormone secretion, augmented the PAP reaction but did not increase the number of positive cells. Similarly, in situ HYB revealed insulin mRNA in ∼5% of the neurons, with no such transcripts identified in glia. We conclude that insulin detected in fetal/neonatal brain extracts is synthesized locally by a select subset of neurons. The biologic function of this neuronal insulin remains to be determined, but possibilities include neurotransmitter activity and/or growth promotion.