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D G Baskin

Publications and source records attributed to D G Baskin.

At least 73 records · Page 4Linked to original sources
PubMed1993

Differential effect of fasting on hypothalamic expression of genes encoding neuropeptide Y, galanin, and glutamic acid decarboxylase.

The effect of caloric deprivation to stimulate hypothalamic neuropeptide Y (NPY) gene expression is hypothesized to represent a physiologically important adaptation in body weight homeostasis. To evaluate the specificity of this response, we used in situ hybridization histochemistry to measure hypothalamic expression of mRNA encoding NPY, galanin, and the two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) in male Wistar rats either fed ad lib or deprived of food for 24 or 48 h. As expected, food deprivation for 24 and 48 h increased preproNPY mRNA levels in the arcuate nucleus by 43 +/- 13% (p = NS) and 127 +/- 29% (p < 0.05 vs. both fed and 24-h fasted groups) when compared to ad lib-fed controls, and hypothalamic preproNPY mRNA levels were significantly correlated to the percent change in body weight over the three groups of rats (r = -0.72; p < 0.05). In contrast, no significant effects of either 24 or 48 h of fasting were observed on hypothalamic levels of preprogalanin, GAD67, or GAD65 mRNA, and no relationship between percent change in body weight and expression of any of these mRNA species could be demonstrated. In conclusion, fasting increases preproNPY mRNA levels in the arcuate nucleus but does not alter expression of other hypothalamic mRNA species pertinent to feeding behavior. This supports the hypothesis that stimulation of NPY gene expression represents an important component of the hypothalamic response to caloric deprivation.

Animals↗
PubMed1993

Selection of oligonucleotide probes for detection of mRNA isoforms.

In this article we discuss strategies for selecting oligonucleotides to target isoform-specific mRNAs, drawing on our experience with isotopically labeled oligonucleotides for ISH of Na,K-ATPase mRNA alpha- and beta-subunit isoforms. Oligonucleotide probes based on one of these isoforms have a high probability of forming nonspecific hybrids with related isoform mRNAs. The design and selection of isoform-specific ISH and how their nucleotide structure influences hybridization are reviewed, as well as basic principles in identifying and evaluating candidate probes. Controls such as Tm analysis and GC content are evaluated. For distinguishing among multiple isoforms of gene families, choose lowest possible homology between isoforms consistent with other factors that influence probe performance.

Animals↗
PubMed1993

Fundamentals of quantitative autoradiography by computer densitometry for in situ hybridization, with emphasis on 33P.

The goal of quantitative autoradiography (QAR) in in situ hybridization (ISH) is to determine the amount of radioactive oligonucleotide or riboprobe present in the corresponding area of the tissue slice that produced an autoradiographic image. This article discusses (a) some of the considerations related to selection and use of computer image analysis systems for accomplishing this objective, (b) development of 14C plastic autoradiographic standards for ISH QAR with 33P, (c) using QAR to develop Tm curves for ISH probes, and (d) measurement of resolution with video imaging systems for QAR.

Animals↗
PubMed1993

Protection from BB rat diabetes by the platelet-activating factor inhibitor BN50730.

The platelet-activating factor inhibitor BN50730, a hetrazepine, was injected intraperitoneally daily from 30 days of age into diabetes-prone BB rats. While 96% (22/23) Tween 80 injected control rats developed diabetes, 0.05 mg/kg BN50730 decreased the frequency to 72% (17/24; n.s.) and 0.5 mg/kg to 56% (14/25; p < 0.01). Mean onset age in controls was 81 +/- 9 days (mean +/- SD), but BN50730 delayed onset to 87 +/- 15 days in the low and 93 +/- 12. days (p < 0.01) in high dose rats. The relative degree of insulitis was reduced in both low (p < 0.01) and high (p < 0.05) dose treated groups. Serum insulin in young prediabetic controls decreased from 84 +/- 34 microU/ml to 38 +/- 20 in the 22 rats developing diabetes (p < 0.001). Serum insulin in BN50730-protected compared to unprotected rats was 114 +/- 49 and 32 +/- 22 (p < 0.001) in the low, and 91 +/- 46 and 21 +/- 15 (p < 0.001) microU/ml in the high dose group, respectively. Increased serum insulin correlated with preserved islet beta cells and decreased insulitis. Treatment did not affect thyroiditis. Thus, platelet-activating factor may be involved in insulitis pathogenesis and platelet-activating factor inhibitors may decrease autoimmune beta cell destruction.

Age Factors↗
PubMed1992

[3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

Animals↗
PubMed1992

Inhibition of hypothalamic neuropeptide Y gene expression by insulin.

Insulin acts in the brain to suppress feeding, whereas neuropeptide Y (NPY) has the opposite effect. Since fasting lowers plasma insulin levels and increases hypothalamic synthesis of NPY, we proposed that insulin may inhibit hypothalamic NPY gene expression. To test this hypothesis, we used RIA and in situ hybridization histochemistry to determine if centrally administered insulin could reduce levels of both NPY and its messenger RNA (mRNA) in discreet hypothalamic regions during fasting. Three groups of Long-Evans rats were entered into a 72-h study protocol. One group was fed ad libitum during this period, while the others were fasted. Fed rats received intracerebroventricular (icv) injections of saline vehicle at 12-h intervals, whereas fasted groups received icv vehicle alone or with insulin (4 mU/12 h). In vehicle-only treated rats, fasting significantly increased expression of preproNPY mRNA in the arcuate nucleus to 179 +/- 20% of fed controls. Administration of icv insulin during fasting abolished this increase (99 +/- 14% of fed controls; P less than 0.05 vs. fasted, vehicle-treated rats). Central insulin administration during fasting also reduced immunoreactive NPY concentrations in samples punched from the paraventricular nucleus (PVN) (875 +/- 122 pg/punch) to levels below vehicle-only treated rats (1396 +/- 435 pg/punch; P less than 0.05), similar to free-feeding control values (814 +/- 170 pg/punch). By comparison, neither fasting nor central insulin administration altered NPY levels in four other hypothalamic regions (supraoptic, ventromedial, dorsomedial, and arcuate nuclei). Continuous icv insulin infusion at a lower dose (2 mU/day) produced a similar result during a shorter period (48 h) of food deprivation in Wistar rats. In this study, central insulin infusion also inhibited the fasting-related increase in arcuate preproNPY mRNA levels and did not affect plasma glucose or insulin levels. This suggests that insulin acts locally to inhibit hypothalamic NPY mRNA expression. We conclude that the increase of levels of NPY in the PVN and preproNPY mRNA in the arcuate nucleus during fasting are inhibited by icv insulin. Fasting, therefore, increases NPY biosynthesis along an arcuate nucleus-PVN pathway in the hypothalamus via a mechanism dependent on low insulin levels.

Animals↗
PubMed1991

Effect of paraformaldehyde fixation on localization and characterization of insulin-like growth factor-I (IGF-I) receptors in the rat brain.

In order to design an approach for localizing IGF-I receptors within the intact CNS, the effect of paraformaldehyde (PAF) fixation on receptor binding was examined. Cryostat sections of rat brains, which were perfused with 0 to 10% PAF, were incubated in 125I-IGF-I and assayed for binding under equilibrium conditions. Binding was quantified from 10 brain regions, involving laminae and nuclei from median eminence, thalamus, hippocampus, choroid plexus, pyriform cortex, and cerebral cortex, by computer densitometry of film autoradiographs. The specific binding, saturation curves, Bmax and Ka, ligand specificity, and binding reversibility of IGF-I binding sites were not significantly affected by 1% or 2% PAF. However, 4% PAF elevated IGF-I receptor total binding, nonspecific binding, and Ka, and decreased Bmax, presumably by increasing the number of tissue-receptor interconnections. Only nonspecific 125I-IGF-I binding persisted when 10% PAF was used. These results indicate that tissue perfused with 2% PAF can be used for localizing IGF-I receptors by autoradiographic binding methods.

Animals↗
PubMed1991

Interleukin-1 beta regulation of islet and thyroid autoimmunity in the BB rat.

Daily injections of high dose human recombinant interleukin-1 beta (IL-1 beta) accelerated the onset of both insulin-dependent diabetes mellitus and lymphocytic thyroiditis in genetically prone BB rats. In diabetes-resistant BB rats, high dose IL-1 beta failed to induce diabetes. Additionally, the presence of neutralizing IL-1 beta antibodies in these rats strongly correlated with inhibition of lymphocytic thyroiditis. Since low dose IL-1 beta protects diabetes-prone rats from IDDM, we conclude that IL-1 beta is a potent modulator of autoimmune diabetes and thyroid disease in genetically susceptible rats.

Adrenal Glands↗
PubMed1991

Central insulin administration reduces neuropeptide Y mRNA expression in the arcuate nucleus of food-deprived lean (Fa/Fa) but not obese (fa/fa) Zucker rats.

By acting in the brain, insulin suppresses food intake, whereas neuropeptide Y (NPY) has the opposite effect. Since fasting increases NPY gene expression in the hypothalamic arcuate nucleus (ARC) and also lowers circulating insulin levels, we hypothesized that the anorexiant effect of insulin could result from insulin inhibition of NPY gene transcription in the ARC. Therefore, we determined whether the administration of insulin (200 mU per 12 hrs) into the 3rd cerebral ventricle of lean (Fa/Fa) female Zucker rats (n = 5) during 48 hrs of food deprivation reduces the expression of preproNPY mRNA in the ARC compared to vehicle-treated controls (n = 5). Coronal sections of rat brain were hybridized with an oligonucleotide probe complementary to preproNPY mRNA and apposed to x-ray film. Hybridization was quantified in both the ARC and the hippocampal dentate gyrus by computerized image analysis of the resulting autoradiographs. Central insulin significantly reduced the area of hybridization in the ARC (0.235 +/- 0.017 mm2; mean +/- SE) compared to vehicle-treated controls (0.331 +/- 0.037 mm2; p less than 0.05), but was without effect in the hippocampus. Thus, insulin reduced the expression of mRNA for NPY specifically in the ARC. Since the genetically obese (fa/fa) Zucker rat is insensitive to the anorexiant effect of insulin and over-expresses NPY in the ARC, we next tested the hypothesis that insulin does not suppress NPY mRNA expression in the ARC of these rats. Consistent with this hypothesis, central insulin administration to obese Zucker rats during 48 hrs of food deprivation (n = 6) did not lower hybridization area in the ARC compared to vehicle alone (n = 4) (0.286 +/- 0.036 vs. 0.248 +/- 0.019 mm2; p greater than 0.05). We conclude that insulin suppresses the expression of mRNA for NPY in the ARC of fasted lean but not obese Zucker rats. Regulation of hypothalamic NPY gene expression by insulin may account for its anorexiant effect, and a defect in this action may contribute to certain forms of obesity.

Animals↗
PubMed1991

Localization of type I insulin-like growth factor receptor messenger RNA in the adult rat brain by in situ hybridization.

Using multiple 35S-labeled oligonucleotide probes concurrently, the type I insulin-like growth factor receptor (IGF-I-R) mRNA was demonstrated by Northern blot hybridization in newborn and adult rat brain as a single species of approximately 11 kilobases. The probes were used to localize IGF-I-R mRNA by in situ hybridization in slices of adult rat brain. The highest levels of IGF-I-R mRNA expression were found in the glomerular and mitral cell body layers of the olfactory bulb, the granule cell body layers of the dentate gyrus and cerebellum, the pyramidal cell body layers of the piriform cortex and Ammon's horn, and the choroid plexus. The lowest levels of IGF-I-R mRNA expression were found in white matter. At the cellular level, IGF-I-R mRNA was expressed by a variety of neurons, by epithelial cells of the choroid plexus, and by ependymal cells of the third ventricle. Of the neuron types studied, the highest levels of IGF-I-R mRNA were consistently found in perikarya of mitral and tufted cells in the olfactory bulb, in pyramidal cells of the piriform cortex and Ammon's horn, and in granule cells of the dentate gyrus. There was a close congruency between the distribution of IGF-I binding and IGF-I-R mRNA at the regional level. Neuropil layers in the cerebral cortex, olfactory bulb, hippocampus, and cerebellum contained a high level of IGF-I binding, whereas the adjacent cell body layers contained a high level of the IGF-I-R mRNA. We conclude that in these regions, IGF-I-R mRNA is synthesized in neuronal cell bodies, and the receptors are transported to axons and dendrites in adjacent synapse-rich layers, where appropriate IGF effects are achieved.

Animals↗
PubMed1991

Effects of ginkgolide B, a platelet-activating factor inhibitor on insulitis in the spontaneously diabetic BB rat.

The BB rat spontaneously develops insulin-dependent diabetes mellitus (IDDM) in association with marked insulitis in the islet of Langerhans. Since platelet-activating factor (PAF-acether) is involved in allergic and inflammatory reactions, we tested a PAF antagonist, Ginkgolide B (BN 52021) for potential effects on islet inflammation and diabetes. Diabetes prone BB/Wor rats were treated daily from weaning at 25 days until 105 days of age with either saline (n = 30, controls), 10 (n = 25, low dose) or 20 (n = 30, high dose) mg/kg body weight of BN 52021. The overall incidence of IDDM was unaffected by treatment. Quantitative analysis of insulin area showed a dose-dependent protection of beta cells by Ginkgolide B, reflected in a 6- (low dose) to 8-fold (high dose) (P less than 0.01-0.005) increase in the insulin/glucagon cell ratio compared to the saline treated rats. Ginkgolide B reduced severe insulitis from 84% in the saline rats developing IDDM to 59% (n.s.) in the low and to 33% (P less than 0.001) in the high dose group. These data suggest that PAF inhibitors may prove useful in immunomodulator therapy of IDDM since beta cells are preserved.

Animals↗
PubMed1990

IL-1 beta modulation of spontaneous autoimmune diabetes and thyroiditis in the BB rat.

Long term effects of in vivo treatment with human rIL-1 beta on diabetogenesis and thyroid disease were determined in the Biobreeding rat. Administration of high dose (10 micrograms/kg) IL-1 beta accelerated the onset of insulin-dependent diabetes mellitus compared to saline-injected controls. High dose treatment resulted in goiter development, pronounced LT, reduced serum T4 levels, and overall growth reduction. In contrast, low dose IL-1 beta (0.5 microgram/kg) administration significantly reduced the frequency of insulin-dependent diabetes mellitus (48%) compared to placebo (86%) and high dose IL-1 beta (93%) treatment groups. Rats protected by low dose IL-1 beta had unaffected growth rates and minimal to no pancreatic and thyroid pathology. Our results demonstrate that exogenous administration of IL-1 beta modulates Biobreeding rat idiopathic autoimmune diabetes and thyroid disease in a dose-dependent manner.

Animals↗
PubMed1990

Distribution of somatostatin-14 and somatostatin-28 gastrointestinal-pancreatic cells of rats and humans.

Somatostatin-14 and somatostatin-28 are biologically active peptides derived from the posttranslational cleavage of prosomatostatin. Because both peptides are found in variable concentrations in the gastrointestinal (GI) tract and pancreas, it has been contended that somatostatin-28 is either an intermediate in the processing to somatostatin-14 or a terminal product derived from prosomatostatin. To address this question, two antisera were used to recognize epitopes in two regions of somatostatin-14; one with high specificity for somatostatin-14 and the other interacting with prosomatostatin, somatostatin-28, and somatostatin-14. Distribution of these peptides was measured in extracts of pancreas and mucosa and submucosa/muscularis from the rat and human GI mucosal biopsies; the antisera were used to immunostain cells in these tissues. Extracts of human and rat intestinal mucosa contained both somatostatin-28 and somatostatin-14. By immunocytochemistry, D cells in stomach and pancreas and neural processes in the intestine, extending into the mucosal villi adjacent to endocrine cells, stained with both antisera indicating the presence of somatostatin-14, prosomatostatin, and possibly somatostatin-28. In contrast, endocrine cells in the gut reacting with antisera against somatostatin-28 did not immunostain with somatostatin-14-specific antisera. Thus, these data suggest that somatostatin-28 is the terminal peptide processed from prosomatostatin in intestinal mucosal cells, whereas somatostatin-14 is the major final product in gastric and pancreatic D cells and neurons. The localization of somatostatin-28 and somatostatin-14 in different cells in the pancreas and GI tract implies that they serve different functions.

Animals↗
PubMed1990

Insulin binding to individual rat skeletal muscles.

Studies of insulin binding to skeletal muscle, performed using sarcolemmal membrane preparations or whole muscle incubations of mixed muscle or typical red (soleus, psoas) or white [extensor digitorum longus (EDL), gastrocnemius] muscle, have suggested that red muscle binds more insulin than white muscle. We have evaluated this hypothesis using cryostat sections of unfixed tissue to measure insulin binding in a broad range of skeletal muscles; many were of similar fiber-type profiles. Insulin binding per square millimeter of skeletal muscle slice was measured by autoradiography and computer-assisted densitometry. We found a 4.5-fold range in specific insulin tracer binding, with heart and predominantly slow-twitch oxidative muscles (SO) at the high end and the predominantly fast-twitch glycolytic (FG) muscles at the low end of the range. This pattern reflects insulin sensitivity. Evaluation of displacement curves for insulin binding yielded linear Scatchard plots. The dissociation constants varied over a ninefold range (0.26-2.06 nM). Binding capacity varied from 12.2 to 82.7 fmol/mm2. Neither binding parameter was correlated with fiber type or insulin sensitivity; e.g., among three muscles of similar fiber-type profile, the EDL had high numbers of low-affinity binding sites, whereas the quadriceps had low numbers of high-affinity sites. In summary, considerable heterogeneity in insulin binding was found among hindlimb muscles of the rat, which can be attributed to heterogeneity in binding affinities and the numbers of binding sites. It can be concluded that a given fiber type is not uniquely associated with a set of insulin binding parameters that result in high or low binding.

Animals↗
PubMed1990

Localization of 3H-prazosin binding sites in the supraoptic and paraventricular nuclei of the human hypothalamus.

Using 3H-prazosin, we have examined the distribution of alpha 1-adrenergic receptors in the supraoptic and paraventricular nuclei of the human hypothalamus. Our studies show that binding sites for 3H-prazosin in human hypothalamus possess pharmacological characteristics similar to those of rat brain. Autoradiographic studies revealed discrete localization of 3H-prazosin to the paraventricular and supraoptic nuclei.

Adult↗