Studies on the presence of angiotensin II in rat brain

Abstract: Angiotensin II-like immunoreactivity was extracted from brains of bilaterally nephrectomized rats with several different extraction procedures (90% methanol, distilled water, 6 M urea, 0.1 N HCI, and 2 M acetic acid). The activity was measured with radioimmunoassays using three different antisera, two of which had been used previously for immunocytochemical studies. With none of the extraction procedures or antisera employed was more than 80 pg/g wet weight of angiotensin II-like immunoreactivity found. Analysis was undertaken with two different reverse-phase high-pressure liquid chromatography systems; in one of these the immunoreactivity did not coelute with angiotensin II or III. On the basis of its elution pattern from a molecular sieving column, the immunoreactivity seems to have a higher molecular weight than angiotensin II. It is concluded that neurons in the brain do not synthesize and store angiotensin II.     

Effects of insulin on rat brain noradrenaline

A single intracardial injection of streptozotocin produced a significant increase in rat hypothalamic noradrenaline while no changes were observed in the olfactory tubercles. The parenteral administration of a single dose of insulin decreased rat hypothalamic noradrenaline; the effect had a rapid onset and lasted for at least six hours. Similar noradrenaline reductions were observed in the olfactory tubercles but in this tissue the depletion started later and recovered earlier. In addition, in olfactory tubercles after insulin injection, tyrosine level and dopamine metabolism were increased. The results show that the increases in hypothalamic NA observed in streptozotocin diabetic rats are counteracted by insulin administration and possibly the consequence of changes in noradrenaline turnover.     

Studies on lipid peroxidation in the rat brain

The aim of this study was to set up a simple procedure for assessing lipid peroxidation (L.P.) and testing the activity of antioxidant compounds. L. P. was determined in rat brain homogenates by measuring the endogenous and stimulated accumulation of malonaldehyde (MDA). MDA was assayed by an HPLC method. Homogenates spontaneously formed appreciable amounts of MDA. The addition of increasing concentrations of FeCl₂ resulted in a linear accumulation of MDA, up to 16.6-fold at 50 M. An organic form of iron (Fe-saccharate) was less active on MDA formation (11.4-fold increase at 100 M). The addition of xanthine-xanthine oxidase resulted in only a 2.4-fold increase in MDA formation. Various antioxidant or chelating compounds effectively inhibited L.P., with IC₅₀ between 0.1 M (phenoxazine) and 4–50 M (-tocopherol). Their potencies depended on the iron concentration and time of preincubation with the homogenates. In conclusion, this is a simple and reliable procedure for studying L.P. and inhibiting agents, provided that the experimental conditions are carefully assessed.     

Effect of insulin on the pyruvate dehydrogenase complex in the rat brain

The level of PDHa and PDHt is substantially reduced in the rat brain 24 hours after alloxan administration. Effects are almost completely reversed by insulin administration. PDHa and PDHt from alloxan rat brains are remarkably activated when assayed on samples obtained by combining and preincubating at 30 degrees C for 30 min a homogenate from fresh unfrozen brains of alloxan rats, with a similarly treated preparation from fresh unfrozen brains of normal or insulin rats. On the contrary, no activation at all is obtained if the preincubation is carried out on homogenates from frozen and thawed brains. In alloxan rats, brain acetyl CoA level decreases remarkably whereas plasma free fatty acid concentration increases. Such changes disappear after insulin administration. The oxygen uptake, the respiratory control index and the ADP/O ratio in mitochondrial preparations obtained from brains of alloxan rats show no modifications at all.     

Studies of aluminum in rat brain

The effects of high aluminum concentrations in rat brain were studied using¹⁴C autoradiography to measure the uptake of [¹⁴C]2deoxy-d-glucose ([¹⁴C]2dG) and microbeam proteon-induced X-ray emission (microPIXE) with a 20-μm resolution to measure concentrations of magnesium, aluminum, potassium, and calcium. The aluminum was introduced intracisternally in the form of aluminum tartrate (Al-T), and control animals were given sodium tartrate (Na-T). The¹⁴C was administered intravenously. The animals receiving Al-T developed seizure disorders and had pathological changes, which included cerebral cortical atrophy. The results showed that there was a decreased uptake of [¹⁴C]2dG in cortical regions in which increased aluminum levels were measured, i.e., there was a correlation between the aluminum in the rat brain and decreased brain glucose metabolism. A minimum detection limit of about 16 ppm (mass fraction) or 3×10⁹ Al atoms was obtained for Al under the conditions employed.     

Studies on the molecular organization of rat insulin secretory granules

Secretory granule-enriched fractions prepared from isolated rat islets of Langerhans, previously labeled in culture for 18 h with [3H]leucine, have been lysed and separated into pH 5.4 soluble and insoluble fractions by zonal sucrose gradient centrifugation. A high proportion of both labeled and immunoreactive rat insulins I and II were recovered in the insoluble granule core fraction in the expected ratio of approximately 60/40, respectively. Essentially equivalent amounts of the rat C-peptides on a molar basis were recovered in the granule supernatant fractions. The proportion of labeled proinsulin in the granule core fraction was less than 2% relative to insulin, while the soluble fraction contained about 8%, which probably arose mainly from disrupted proinsulin-rich noncrystalline prosecretory vesicles. Electron microscopic examination of the granule core fraction revealed large numbers of well preserved crystalline cores exhibiting typical dimensions and regular internal spacings of normal mature rat beta-granule inclusions. These results provide direct biochemical evidence that the beta-granules are nonuniform in composition with the insulin contained mainly in a crystalline state in the electron-dense central inclusions while the C-peptide is dissolved in the fluid bathing the crystalline hormone. The significance of this structural organization of the beta-granule is discussed.     

Studies on the origin of choline in the brain of the rat

1. Labelled precursors of choline, namely ethanolamine, dimethylaminoethanol and methionine and also labelled choline itself were injected intraperitoneally into the adult female rat and the incorporation into lipids and water-soluble fractions was traced in liver, blood and brain. 2. No significant free choline was detected and no labelling of the phosphorylcholine of blood. There was, however, considerable labelling of the phosphorylcholine of brain and liver. 3. After intracerebral injection, [1,2-(14)C]dimethylaminoethanol was rapidly phosphorylated and converted into phosphatidyldimethylaminoethanol, presumably by the cytidine pathway. 4. In view of the pattern of labelling and the amount of phosphatidylcholine in the tissues examined, it seems highly likely that choline is transported to the brain by the blood in a lipid-bound form.     

Melatonin enhances leptin expression by rat adipocytes in the presence of insulin

Leptin and melatonin play an important role in the regulation of body mass and energy balance. Both hormones show a circadian rhythm, with increasing values at night. In addition, melatonin receptors were recently described in adipocytes, where leptin is synthesized. Here, we investigated the influence of melatonin and its interaction with insulin and dexamethasone on leptin expression. Isolated rat adipocytes were incubated with melatonin (1 nM) alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) for 6 h. Melatonin or insulin alone did not affect leptin expression, but together they increased it by 120%. Dexamethasone increased leptin mRNA content (105%), and this effect was not enhanced by melatonin. Simultaneous treatment with the three hormones provoked a further increase in leptin release (250%) and leptin mRNA (100%). Melatonin prevented the forskolin-induced inhibition (95%) of leptin expression. In addition, melatonin's ability to stimulate leptin release (in the presence of insulin) was completely blocked by pertussis toxin and luzindole. To gain further insight into the molecular basis of melatonin and insulin synergism, the insulin-signaling pathway was investigated. Melatonin increased the insulin-induced insulin receptor-beta tyrosine phosphorylation, which led to an increased serine phosphorylation of the downstream convergent protein Akt. We concluded that melatonin interacts with insulin and upregulates insulin-stimulated leptin expression. These effects are caused by melatonin binding to the pertussis toxin-sensitive G(i) protein-coupled membrane receptor (MT1 subtype) and the cross talk with insulin, since insulin receptor and its convergent target Akt are coactivated by melatonin.     

Evidence for the presence of diacylglycerol kinase in rat brain myelin

The previous demonstration that incubation of brain slices with [³²P]phosphate brings about rapid tabeling of phosphatidic acid in myelin suggests that the enzyme involved should be present in this specialized membrane. DAG kinase (ATP:1,2-diacyglycerol 3-phosphotransferase, E.C. 2.7.1.107) is present in rat brain homogenate at a specific activity of 2.5 nmol phosphatidic acid formed/min/mg protein, while highly purified myelin had a much lower specific activity (0.29 nmol/min/mg protein). Nevertheless, the enzyme appears to be intrinsic to this membrane since it can not be removed by washing with a variety of detergents or chelating agents, and it could not be accounted for as contamination by another subcellular fraction. Production of endogenous, membrane-associated, diacylglycerol (DAG) by PLC (phospholipase C) treatment brought about translocation from soluble to particulate fractions, including myelin. Another level of control of activity involves inactivation by phosphorylation; a 10 min incubation of brain homogenate with ATP resulted in a large decrease in DAG kinase activity in soluble, particulate and myelin fractions.     

Studies on sodium transport in rat brain nerve-ending particles

(1) Nerve-ending particles isolated from crude mitochondrial preparations from rat brain by discontinuous Ficoll density gradient ultracentrifugation were shown to possess a Mg2+ and energy-dependent transport system for Na+. (2) Ouabain or iodoacetate plus cyanide exerted an inhibitory effect on the outflux but not the influx of Na+. (3) When K+ was added to a medium containing particles loaded with Na+ (22Na), an immediate release of Na+ from these particles was observed; this suggests the existence of a Na+-K+ exchange transport system. (4) The K+ effect was inhibited by 10(-4) M-ouabain only at low (about 3.3 mM), but not at high (20 mM), K+ concentrations. (5) The uptake and release of Na+ by the nerve-ending particles were found to be temperature-dependent. (6) Only nerve-ending particles with intact synaptosomal membranes were found to transport Na+ actively.     

Studies on the binding of nucleotides by rat brain hexokinase

Various nucleoside di- and triphosphates have been compared with respect to their ability to protect rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activity against inactivation by chymotrypsin, glutaraldehyde, heat, and 5,5′-dithiobis(2-nitrobenzoic) acid. ATP could be distinguished from other nucleoside triphosphates in these comparisons, which may be related to the specificity with which ATP is utilized as a substrate. All nucleoside derivatives examined provided substantial protection against two or more of the above inactivating agents, indicating relatively nonspecific binding of nucleotides by brain hexokinase, consistent with a similar lack of specificity in the inhibition of this enzyme by nucleoside derivatives. The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tetraiodofluorescein (TIF) was enhanced by binding to brain hexokinase. TNS binding was not affected by the presence of various relevant metabolites (Glc, glucose 6-phosphate, ATP), nor did TNS inhibit the enzyme. In contrast, substantial (approximately 70%) decreases in the fluorescence of bound TIF resulted from the addition of various nucleoside derivatives, and TIF served as a competitive inhibitor of brain hexokinase. These observations are consistent with the view that TIF binds to a nucleotide binding site of the enzyme. The inability of nucleotides to totally displace TIF was taken to indicate the existence of an additional TIF binding site (or sites) discrete from the catalytic site, and probably identical to the site(s) at which TNS binds with no effect on catalytic activity. The effects of saturating levels of ATP and ADP were not additive indicating that both compounds were displacing TIF from the same site i.e., a common nucleotide binding site. Glc, mannose, and 2-deoxyglucose greatly enhanced the ability of nucleotides to displace TIF, while fructose, galactose, and N-acetylglucosamine did not, indicating the existence of interactions between hexose and nucleotide binding sites; the hexoses themselves were not effective at displacing TIF. The enhanced binding of nucleotides in the presence of the first three hexoses but not the latter three can be directly correlated with the relative ability of these hexoses to induce specific conformational changes in the enzyme. The hexoses themselves were not effective at displacing TIF. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate could also displace TIF, and as with the nucleotides, a maximum of approximately 70% decrease in fluorescence was observed and the effectiveness of glucose 6-phosphate was enhanced in the presence of Glc. Other hexose 6-phosphates tested were not effective at displacing TIF. The specificity with which hexose 6-phosphates displaced TIF could be correlated with their ability to induce specific conformational change in the enzyme. The results are discussed as they relate to the kinetic mechanism and allosteric regulation by nucleotides that have been proposed for this enzyme.     

Studies on the subsite specificity of the rat brain puromycin-sensitive aminopeptidase

The specificity of the puromycin-sensitive aminopeptidase from rat brain was examined. Using L-alanyl-beta-naphthylamide as substrate Vmax of the reaction was shown to be pH independent over the range of 5.5-9.0, while Km exhibited a pKa of 7.7. This latter value corresponds to the pKa of the amino group of the substrate. Using X-Ala and X-Leu to examine the specificity of the P1 site it was found that Arg and Lys exhibit the highest affinity, followed by Met, Val, Leu, Trp, and Phe, which bind congruent to 5- to 20-fold less well. Although Km varied more than 20-fold within this series, Vmax showed considerably less variation. Significantly weaker binding was observed with a P1 Gly, Ala, Ser, or Pro with no binding detectable with a P1 Glu. The presence of a P'1 Leu compared to P'1 Ala results in an approximate 10-fold decrease in Km with little change in Vmax. The effect of varying P'1 residues was examined with the series Leu-X. In this case basic and hydrophobic amino acids, with the exception of Val, all exhibit nearly the same Km. The binding of Arg-Arg and Lys-Lys showed the same Km as obtained for Arg-Leu or Lys-Leu, respectively. When Leu-Ser-Phe was compared to Leu-Ser the P'2 residue led to a 100-fold decrease in Km and slightly less than a 5-fold increase in Vmax. In contrast the addition of a P'2 Met to Leu-Trp results in only a 3-fold decrease in Km and a 3-fold increase in Vmax. The results indicate a preference for a basic or hydrophobic residue in the P1 and P'1 sites and indicate subsite-subsite interactions which primarily affect binding.     

Studies with antipeptide antibody suggest the presence of at least two types of glucose transporter in rat brain and adipocyte

Three antipeptide antibodies were prepared by immunizing rabbits with synthesized short peptides corresponding to residues 215-226, 466-479, and 478-492 predicted from the cDNA of both the human hepatoma HepG2 and rat brain glucose transporters. All three antibodies were found to precipitate quantitatively the [3H]cytochalasin B photoaffinity-labeled human erythrocyte glucose transporter. Each antibody also recognized the rat brain protein of Mr 45,000 on immunoblots, and a similar molecular weight protein was labeled with [3H]cytochalasin B in a D-glucose-inhibitable manner, suggesting that this protein is glucose transporter. However, only up to 30% of the labeled rat brain glucose transporters were precipitated, even by repeated rounds of immunoprecipitation. In addition, these antibodies were observed to be unable to immunoprecipitate significantly the [3H]cytochalasin B-labeled rat adipocyte glucose transporter. Further, one-dimensional peptide maps of [3H]cytochalasin B-labeled human erythrocyte and adipocyte glucose transporters generated distinct tryptic fragments. Although Mr 45,000 protein in rat adipocyte low density microsomes was detected on immunoblots and its amount was decreased in insulin-treated cells, the rat adipocyte low density microsomes were much less reactive on immunoblots than the rat brain membranes in spite of the fact that the rat adipocyte low density microsomes contained more [3H]cytochalasin B-labeled glucose transporters. In addition, the ratio of cytochalasin B-labeled glucose transporter per unit HepG2-type glucose transporter mRNA was more than 10-fold higher in rat adipocyte than in rat brain. These results indicate that virtually all the human erythrocyte glucose transporters are of the HepG2 type, whereas this type of glucose transporter constitutes only approximately 30 and 3% of all the glucose transporters present in rat brain and rat adipocyte, respectively; and the rest, of similar molecular weight, is expressed by a different gene.     

Evidence for the presence of a glycosphingolipid-transfer protein in rat brain cytosol

Proteins in the postmicrosomal supernatant fraction of rat brain catalyzed the transfer of bovine brain galactocerebroside, sulfatide, and ganglioside GM1 from unilamellar liposomes to the rat erythrocytes or ghosts. The vesicles were made with egg yolk lecithin, cholesterol, 3H-labelled glycolipid, and a trace of [14C]triolein as a nonexchangeable marker. The routine assay of the glycosphingolipid transfer consisted of incubation of the donor liposomes with erythrocytes in the presence or absence of supernatant protein in physiological buffer at 37 degrees C for various time intervals. After the incubation, the erythrocytes were separated from the vesicles by centrifugation and the extent of protein-catalyzed transfer of labelled glycolipid in the membrane-bound total lipid fraction was determined by scintillation spectrometry. The fraction of [3H]glycosphingolipid transferred is represented by a change in the 3H/14C ratios at initial and subsequent time intervals. The glycosphingolipid transfer catalyzed by the supernatant protein was found to be logarithmic, whereas the protein-independent transfer was linear over a period of 3-4 h. The rate constant (K) and half time (t1/2) of the protein-catalyzed transfer reaction of cerebrosides and sulfatides were almost the same, while the transfer of ganglioside GM1 occurred at a slightly faster rate, probably owing to the greater aqueous solubility of this lipid. The transfer activity was also increased in a manner dependent on the amount of supernatant protein added up to 10 mg. The catalytic activity of the protein was lost when heated at 70 degrees C for 5 min. The pH optimum of the activity was around 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of rat brain insulin receptor kinase activity in diabetes

Insulin receptors from rat brain regions were studied for insulin binding and receptor associated kinase activity, in alloxan induced short-term and long-term diabetes, and insulin induced hypoglycemia. Insulin receptor activity was assessed by [¹²³I]insulin binding, and basal as well as insulin stimulated kinase activity of the receptor, expressed as phosphorylation of the synthetic peptide poly (Glu-Tyr (4:1)). Regional distribution pattern elicited the highest binding and kinase activity in the olfactory bulb. Diabetes caused a significant increase in the kinase activity. The data suggests that brain insulin receptor kinase is regulated differently compared to peripheral tissues and supports the concept of an active brain insulin receptor in vivo.     

Radioautographic and quantitative study of insulin binding sites in the rat brain

In the present study, we describe the specificity and the autoradiographic distribution of insulin binding sites in the rat central nervous system (CNS) after in vitro incubation of brain sections with [125I]-14A insulin. Increasing concentrations of unlabeled insulin produced a dose-dependent inhibition of [125I]-insulin binding which represented 92 +/- 2% displacement with 3 X 10(-5) M, whatever the brain sections tested. Half-maximum inhibition with native insulin was obtained with 2.2 X 10(-9) M, with 10(-7) M proinsulin whereas glucagon had no effect. Under our experimental conditions, no degradation of [125I]-insulin was observed. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed a widespread distribution of [125I]-insulin in rat CNS. However, quantitative analysis of the autoradiograms with 10(-10) M of labeled insulin, showed a high number of [125I]-insulin binding sites in the choroid plexus, olfactory areas, in both cerebral and cerebellar cortices, the amygdaloid complex and in the septum. In the hippocampal formation, the dorsal dentate gyrus and various subfields of CA1, CA2 and CA3 were labeled. Moreover, arcuate, dorso- and ventromedial nuclei of the hypothalamus contained high concentrations of [125I]-insulin whereas a low density was observed in the mesencephalon. The metabolic role of insulin in the CNS is supported by the large distribution of insulin binding sites in the rat brain. However, the presence of high affinity binding sites in selective areas involved in perception and integrative processes as well as in the regulation of both feeding behavior and neuroendocrine functions, suggests a neuromodulatory role of insulin in the brain.