Identification of insulin in rat brain.

Insulin concentrations in acid/ethanol extracts of the whole rat brain were on the average 25 times higher than plasma insulin levels. Brain insulin was indistinguishable from authentic pancreatic insulin, based on its behavior in radioimmunoassay, radioreceptor assay, and bioassay and its chromatographic pattern on Sephadex G-50 column chromatography. Insulin was found in all regions of the brain examined, but distribution was uneven. Some regions had insulin concentrations as much as 100 times higher than in plasma; levels at least 10 times higher were found in other regions. The role of insulin in the central nervous system is not clear at present but, because both insulin and insulin receptors are abundant in the central nervous system, an extensive physiological regulation of the central nervous system by insulin is proposed.     

Evidence for preserved insulin responsiveness in the aging rat brain

GeroScience - Insulin appears to exert salutary effects in the central nervous system (CNS). Thus, brain insulin resistance has been proposed to play a role in brain aging and dementia but is...     

Regional distribution of 11 beta-hydroxysteroid dehydrogenase in rat brain

The activity and distribution of 11 beta-hydroxysteroid dehydrogenase in rat brain is described. Oxidation of corticosterone to 11-dehydrocorticosterone was significantly increased by NADP; the reverse reaction was increased by NADPH. Cortisol was a poor substrate. Both 11-dehydrogenase (11-DH) and 11-oxoreductase (11-OR) activities were found in brains of rats from 6 days to adult, and 11-DH and 11-OR activities were positively correlated with each other. Highest enzyme activities were found in pituitary, cerebellum, hippocampus, and cortex. Lower levels were found in the olfactory region, hypothalamus, brain stem, preoptic nucleus, and amygdala. Two antisera to 11-DH, designated 56-125 and 56-126, reacted with a 34K component corresponding in mass to rat liver 11-DH on Western blots. The dominant species of protein in all brain regions reacting with rat liver 11-DH antibody 56-125, was at 26K mol wt. Antiserum 56-126 did not cross-react with the 26K protein. The 26K component was not a 34K degradation product. In each region of the brain, Western blot analysis showed that the 26K band intensity was directly proportional to enzyme activity. However, the 26K protein was devoid of 11-DH activity. All 11-DH and 11-OR activities were associated with the 34K antigen. The data demonstrate the nonuniform distribution of 11-DH in brain tissue. They are consistent with the notion that 11-DH may confer upon brain the ability to control intracellular levels of active glucocorticoids and in this way mediate steroid function within the cell.     

Regional analysis of rat brain proteins during senescence

Brains of male Fischer-344 rats aged 3-4 months and 28-30 months were dissected into 11 regions, and the patterns of brain proteins in these regions were analyzed using two dimensional gel electrophoresis in conjunction with a sensitive silver stain detection method. Several hundred abundant and moderately abundant brain proteins were detected in each region using this method. At 3-4 months, most proteins were present in approximately equal amounts in each of the 11 regions. On the whole, this distribution was maintained as a function of age. One protein of 21 kdal pI 5.1 was present in the cerebellum in greater amount than the other regions at both 3-4 months and 28-30 months. Two proteins, 44 kdal pI 5.4 and 47 kdal pI 5.2 were present at increased levels in the inferior colliculus of 28-30 month animals compared to 3-4 month animals. Of the abundant and moderately abundant brain proteins detected by this method, there were none which showed major decreases in levels as a function of age. These results provide support for the concept that the molecular mechanisms which result in differential gene expression in different regions of the young adult rat brain are operative and are maintained in the brains of senescent rats.     

Sodium at high concentrations increases glucose-induced insulin secretion in the perfused rat pancreas

To study the effects of supraphysiologically high sodium concentrations on insulin secretion, the rat pancreas was perfused with normal Krebs-Ringer bicarbonate buffer (KRBB) solution and with KRBB solutions containing an additional 25 and 50 mmol/l NaCl. The first phase of insulin secretion in response to 18 mmol/l glucose was enhanced in a dose-dependent manner, from 20.8±4.0 pmol for 4 min in normal KRBB to 36.2±10.3 pmol (P<0.05) and 60.3±14.6 pmol (P<0.01) with addition of 25 and 50 mmol/l NaCl, respectively. The second phase secretion was significantly increased by the addition of 50 mmol/l NaCl (259.4±38.6 vs 170.9 ±47.4 pmol for 16 min P<0.05). The addition of 50 mmol/l isethionate Na to normal KRBB solution also increased the first and second phases of insulin secretion in response to glucose. The increase in osmolarity by the addition of mannitol to normal KRBB solution did not affect the glucose-induced insulin secretion. A high sodium concentration affected neither tolbutamide-induced nor arginine-induced insulin secretion. It is suggested that sodium concentrations have an important role in insulin secretion in response to glucose, but not to other secretagogues. Received: 29 January 1998 / Accepted in revised form: 24 June 1998     

Stimulation of ornithine decarboxylase activity by insulin in developing rat brain

Insulin administered ip or intracisternally (ic) increased the activity of ornithine decarboxylase (ODC) in whole brains and brain parts of neonatal rats. Maximal stimulation of activity occurred 4-5 h after ip administration. At the highest doses, insulin stimulated ODC activity by up to 5- and 8-fold after ip and ic injection, respectively. The same amount of insulin given ic caused greater increases in activity than when given ip. Insulin stimulated ODC activity in 2-day-old and in 17- to 60-day-old rats but not in 5- or 9-day old neonates or 80-day-old adults. When insulin-induced hypoglycemia was prevented by giving dextrose, the stimulation of ODC activity was approximately the same as that in animals receiving insulin without dextrose. This indicates that insulin-induced stimulation of brain ODC activity was not caused by insulin-induced hypoglycemia or physiological responses to hypoglycemia. Since ODC is considered an indicator of growth stimulation, these results suggest that insulin or insulin-like peptides have a role in the regulation of brain development.     

Regional glucose metabolism and glutamatergic neurotransmission in rat brain in vivo

Multivolume (1)H-[(13)C] NMR spectroscopy in combination with i.v. [1,6-(13)C(2)]glucose infusion was used to detect regional glucose metabolism and glutamatergic neurotransmission in the halothane-anesthetized rat brain at 7 T. The regional information was decomposed into pure cerebral gray matter, white matter, and subcortical structures by means of tissue segmentation based on quantitative T(1) relaxation mapping. The (13)C turnover curves of [4-(13)C]glutamate, [4-(13)C]glutamine, and [3-(13)C]glutamate + glutamine were fitted with a two-compartment neuronal-astroglial metabolic model. The neuronal tricarboxylic acid cycle fluxes in cerebral gray matter, white matter, and subcortex were 0.79 +/- 0.15, 0.20 +/- 0.11, and 0.42 +/- 0.09 micromol/min per g, respectively. The glutamate-glutamine neurotransmitter cycle fluxes in cerebral gray matter, white matter, and subcortex were 0.31 +/- 0.07, 0.02 +/- 0.04, and 0.18 +/- 0.12 micromol/min per g, respectively. The exchange rate between the mitochondrial and cytosolic metabolite pools was fast relative to the neuronal tricarboxylic acid cycle flux for all cerebral tissue types.     

Regional brain concentrations of calcitonin gene-related peptide in spontaneously hypertensive and normotensive Wistar-Kyoto rats

The regional brain and spinal cord concentrations of calcitonin gene-related peptide (CGRP) were measured in age-matched (22-23-week-old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The highest concentration of CGRP in the WKY rats was in the spinal cord (172 +/- 9 pmol/g), followed by the medulla oblongata/pons (88 +/- 5 pmol/g). The relative order of distribution in the remaining regions was: hypothalamus (12.6 +/- 0.8 pmol/g) = striatum greater than thalamus greater than midbrain = hippocampus greater than cortex (2.1 +/- 0.3 pmol/g). The concentration of CGRP in the cerebellum was at the level of the assay's sensitivity (0.5 pmol/g). The relative order of distribution in the SHR strain was essentially the same. However, in comparison with the WKY rats, the SHR had significantly lower levels of CGRP in the hippocampus (-47%), striatum (-49%) and medulla oblongata/pons (-24%), and in the spinal cord (-24%). In younger age-matched (16-17-week-old) rats, the spinal cord and medulla oblongata/pons concentrations of CGRP were also lower in SHR than in WKY rats. CGRP is a putative neurotransmitter which, when administered centrally or peripherally, has potent cardiovascular effects. The reduced levels of this peptide may be an important factor in the cardiovascular and/or behavioural abnormalities of the SHR strain.     

Localization of insulin receptor mRNA in rat brain by in situ hybridization

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.     

Pioglitazone attenuates basal and postprandial insulin concentrations and blood pressure in the spontaneously hypertensive rat

Subjects with hypertension are hyperinsulinemic and resistant to insulin-stimulated glucose uptake. A similar paradigm is found in the spontaneously hypertensive rat (SHR). These findings suggest the possibility that insulin resistance and hyperinsulinemia may play an important role in blood pressure regulation. Pioglitazone, a thiazolidinedione derivative, sensitizes target tissues to insulin and decreases hyperglycemia and hyperinsulinemia in various insulin-resistant animals. The purpose of this study was to assess the influence of pioglitazone administration on pre- and postprandial glucose and insulin concentrations and determine whether changes in β-cell secretion resulted in any change in blood pressure measurements. Twelve SHR were fed custom diets ad libitum, six with and six without pioglitazone (20 mg/kg chow). Fasting and postprandial glucose levels were unaltered by pioglitazone treatment. Fasting insulin concentrations were similar at week 1, but were significantly lower (P < .01) in the pioglitazone group at weeks 3 (1.89 ± 0.3 v7.94 ± 1.5 ng/mL) and 4 (4.5 ± 1.4 v9.1 ± 0.7 ng/mL), compared with the control group. Pioglitazone also significantly (P < .01) lowered postprandial insulin concentrations after an oral glucose challenge. Systolic, mean, and diastolic blood pressures were significantly lower (P < .01), 177 ± 3 v190 ± 4.7 mm Hg, 162 ± 2.1 v175 ± 5.9 mm Hg, and 156 ± 2.1 v168 ± 6.2 mm Hg, respectively, in the animals receiving pioglitazone versus the control group. Heart rate, body weight, serum cholesterol, and triglyceride levels were comparable between the two groups. In conclusion, pioglitazone significantly decreased fasting and postprandial insulin concentrations and effectively lowered blood pressure in the SHR.     

Regional differences of neuron loss of rat brain in old age.

DNA content was measured in four areas of the brain (cerebral hemisphere, cerebellum, diencephalon, and lower brain stem) of young adult (5-month-old) and aged 24- to 30-month-old Long-Evans rats. There was no change in DNA content in all four areas in old age. The number of brain cells was counted in three areas of the brain (cerebral hemisphere, cerebellum and brain stem) with an ultrasonic separation method. In old age, there was significant loss in the total number of brain cells in the cerebellum but no cell loss in the cerebral hemisphere and the brain stem. The neuron number was significantly decreased and the glia/neuron ratio was increased in the cerebellum and cerebral hemisphere, but neither of them changed in the brain stem in old age. The cerebellum showed significantly more neuron loss than the cerebral hemisphere in old age. DNA content in individual brain cells in the cerebral hemisphere, cerebellum and brain stem does not change in old age; thus, calculation of brain cell number from DNA content in the brain of old rats is justified.     

Participation of the 26S proteasome in the regulation of progesterone receptor concentrations in the rat brain

The aim of this study was to investigate the participation of the 26S proteasome in the regulation of progesterone receptor (PR) concentrations in the rat brain in vivo. Ovariectomized adult female rats were treated with estradiol (10 microg/100 g s.c.), estradiol + progesterone (400 microg/100 g), and vehicle (corn oil/10% ethanol) in the presence or absence of the proteasome inhibitor Z-Ile-Glu (OBu(1))-Ala-Leu-H (PSI, 300 microg/100 g). Proteins were extracted from the preoptic area, the hippocampus, and the frontal cortex, and processed for Western blot. Estradiol-induced PR expression in the preoptic area and the hippocampus, whereas progesterone did not modify the effect of estradiol. Neither estradiol nor progesterone modified PR content in the frontal cortex. PSI treatment increased PR content in the preoptic area and the hippocampus. This increase was significant in both regions after 24 h of the treatment with progesterone + PSI in the animals primed with estradiol. In this case, the content of both PR isoforms (PR-A and PR-B) was increased in a similar manner by PSI in the preoptic area (90 and 97%) and in the hippocampus (49 and 50%). PSI did not affect PR content in the frontal cortex. Our results suggest that the 26S proteasome could participate in the turnover of PR in the preoptic area and the hippocampus of the rat in vivo.     

Regional induction of c-fos-like protein in rat brain after estradiol administration.

The protooncogene c-fos is induced in rat brain by various forms of physiological stimulation. In this study immunocytochemical staining for a peptide fragment of the c-fos protein was used to assess estradiol's effects on c-fos in rat brain. After estradiol benzoate (100 micrograms/kg) administration to ovariectomized rats, the number of cells staining for c-fos-like protein increased in the anterior medial preoptic area, the medial preoptic area, the medial amygdala nucleus, and the ventromedial nucleus of hypothalamus, all regions rich in estradiol-concentrating cells. This increase peaked between 12-48 h (depending on the region) after estradiol administration and was not observed in several areas with a lower density of estradiol-concentrating cells. In the most affected region, the anterior medial preoptic area, estradiol effects were dose dependent and not altered by progesterone administration. It is not yet clear whether estradiol induces c-fos expression in brain directly or whether c-fos is part of a cascade of mechanisms by which estradiol regulates gene expression.     

Effects of neonatal administration of monosodium glutamate on four neuropeptide concentrations in the rat brain

The present study was designed to measure concentrations of four neuropeptides in different brain regions in monosodium glutamate(MSG)-treated rats and to assess molecular forms of each peptide with gel and high performance liquid chromatography (HPLC). MSG(4mg/kg body weight) or 10% NaCl was injected subcutaneously on postnatal days 1, 3, 5, 7 and 9 to male littermates which were subsequently used on postnatal day 100. Rats were sacrificed by decapitation, and the brains were dissected into ten discrete regions. The brain extracts were subjected to measurement of four neuropeptides; somatostatin (SRIF), neuropeptide Y (NPY), atrial natriuretic polypeptide(ANP), and a novel pituitary polypeptide 7B2 by specific radioimmunoassays. Significant increase (p less than 0.01) in midbrain SRIF content was observed in MSG-treated rats, though there was no significant change in hypothalamic SRIF content. Significant reduction (p less than 0.05) in hypothalamic NPY content was also found in MSG-treated rats. Hypothalamic ANP content was similar in both MSG-treated and control rats. A significant increase of 7B2 content was found in substantia nigra/ventral tegmentum and hypothalamus (p less than 0.05 or p less than 0.01, respectively) in MSG-treated rats. These four immunoreactivities were further characterized by gel permeation or high pressure liquid chromatography (HPLC). Chromatographic analysis of SRIF immunoreactivity revealed that there were two distinctive peaks and smaller molecular weight component corresponding to SRIF. Fractionation of NPY or 7B2 immunoreactivity by gel permeation showed a single major peak which was identical to the synthetic NPY or 7B2 immunoreactivity from porcine pituitary extract. HPLC analysis for ANP immunoreactivity also showed that the major immunoreactive component corresponded to synthetic rat ANP. MSG treatment could not produce any major alterations in proportions of molecular forms studied. These results suggest that MSG treatment in neonates might produce the alterations in SRIF, NPY and 7B2 content in the discrete brain regions including the hypothalamus.     

Low concentrations of interleukin-1 stimulate and high concentrations inhibit insulin release from isolated rat islets of Langerhans

Isolated rat islets were incubated either with crude, affinity-purified or recombinant human interleukin-1 for 1 to 6 days. A significant (20-60%) increase of insulin release was observed at low concentrations of all three interleukin-1-containing preparations. In contrast, higher concentrations dose-dependently inhibited the insulin release. The increased insulin secretion occurred at concentrations below those necessary to augment the mitogen response to phytohaemagglutinin of murine thymocytes in vitro. These doses (0.05-0.5 U/ml) correspond to 0.2-2 ng of recombinant interleukin-1 per ml, equal to approximately 0.01-0.1 pmol/ml. In doses of 0.6-1.8 U/ml affinity-purified interleukin-1 significantly increased the islet insulin content per ng of DNA, indicating a stimulation of insulin-biosynthesis. The data support the concept that low concentrations of interleukin-1 may play a role in priming the physiological secretion of insulin.     

Estrous cycle variations in cholecystokinin and substance P concentrations in discrete areas of the rat brain

Cholecystokinin (CCK) and substance P (SP) were measured in discrete areas of the rat brain at different stages of the estrous cycle. Significantly higher levels of CCK were found in the lateral septum during diestrus as compared to proestrus. In the parietal cortex, CCK concentrations were significantly higher in diestrus than in proestrus. In the amygdala, estrous levels of CCK were significantly higher than proestrous levels. SP concentrations were significantly higher in diestrus than in proestrus in the medial and lateral septum, and the medial and lateral preoptic area. In the amygdala and ventral tegmental area, SP concentrations were significantly higher in estrus than in proestrus. These data suggest that certain CCK and SP neuronal systems may play a role in regulating the hypothalamo-pituitary-gonadal axis and/or be involved in steroid-dependent behavior.     

Regional dissociation of beta-endorphin and enkephalin contents in rat brain and pituitary.

beta-Endorphin and enkephalin in extracts of whole brain, various brain regions, adenohypophysis, and combined pars intermedia and neurohypophysis of the rat were measured by radioimmunoassay. In brain extracts, the immunoreactive substances were further separated according to molecular size by gel filtration. beta-Endorphin was found in the diencephalon but not in the hippocampus, cerebral cortex, cerebellum, and striatum. Enkephalin was found predominantly in the striatum and diencephalon. Attention is called to possible artifactual interference by myelin basic protein in the immunoassays for beta-endorphin in some regions of the brain. In the pituitary, enkephalin was mainly restricted to the pars intermedia-neurohypophysis. Neither adrenalectomy nor hypophysectomy significantly altered levels of beta-endorphin in brain extracts. Adrenalectomy increased the levels of beta-endorphin in adenohypophysis and pars intermedia-neurohypophysis; after adrenalectomy, enkephalin was also increased in the adenohypophysis but less so in the pars intermedia-neurohypophysis. These results show that brain endorphin levels are independent of pituitary endorphin levels; they suggest that beta-endorphin-containing neurons and those containing enkephalin constitute two separate groups of brain cells.     

Free and total insulin integrated concentrations in insulin dependent diabetes

Studies of the 24 hr insulin concentration profiles in diabetic subjects on chronic exogenous insulin have been hampered by the presence of endogenous anti-insulin antibody, which gives spurious estimates of radioimmunoassayable insulin concentrations. The introduction of polyethylene glycol precipitation of endogenous antibody has allowed development of reliable assays for determiniation of free and total insulin concentration in subjects on insulin therapy. This article reports our observations of plasma free and total insulin concentration in 50 Type I and Type II ambulatory insulin dependent diabetics, utilizing a continuous 24 hr blood withdrawal technique. In response to exogenous insulin, study subjects had marked elevations in insulin concentrations compared to controls. Mean free insulin integrated concentration was 3.5-fold higher in diabetics than nondiabetics. Mean total insulin integrated concentration was 868 μU/ml, more than 20 times in excess of total insulin concentration in nondiabetics. There was a wide range among diabetics in the percentage of total insulin in the free insulin fraction. Neither free nor total insulin integrated concentration correlated with dose of exogenous insulin. Free and total insulin concentration profiles showed a limited range of variation in insulin concentration during the 24 hr of study, no subject having a profile that mimicked that observed in nondiabetic subjects. Glucose integrated concentration showed no correlation with free insulin integrated concentration, however, it did correlate inversely with the percentage of total insulin in the free insulin fraction. These data emphasize the difficulty in establishing normal patterns of insulin among diabetic subjects on conventional subcutaneous insulin therapy.     

Fibrinolysis and atherogenesis in the JCR:LA-cp rat in relation to insulin and triglyceride concentrations in blood

Summary Increased concentrations of plasminogen activator inhibitor type-1 (PAI-1) in blood and attenuated fibrinolytic activity, hypertriglyceridaemia, and insulin resistance are common in subjects with obesity and non-insulin-dependent diabetes mellitus who are at markedly increased risk for coronary artery disease. To clarify potentially causal relationships between these phenomena, we studied JCR:LA-cp rats, animals that are insulin resistant and prone to vasculopathy. Blood and aortas were obtained from lean and corpulent animals at 1, 2, 4, 6, and 9 months of age. The homozygous corpulent rats were hyperinsulinaemic and hypertriglyceridaemic at all ages tested. Increased activity of PAI-1 was present in blood from corpulent animals at 1, 6, and 9 months of age. Positive correlations were observed between blood PAI-1 and both insulin and triglycerides. As judged from results with aortic rings in in vitro culture, the increased PAI-1 in blood was anteceded by increased expression of PAI-1 in arterial walls. Thus, changes indicative of inhibition of the fibrinolytic system capacity precede gross atherosclerosis. [Diabetologia (1998) 41: 141–147] Received: 8 July 1997 and in revised form: 23 September 1997