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D G Baskin

Publications and source records attributed to D G Baskin.

At least 55 records · Page 3Linked to original sources
PubMed1996

Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice.

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 micrograms) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 +/- 1.2 vs. 24.5 +/- 3.8 mmol/l; P < 0.01) and insulin levels (7,263 +/- 1,309 vs. 3,150 +/- 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.

Animals↗
PubMed1995

Selective uptake of radiolabeled annexin V on acute porcine left atrial thrombi.

BACKGROUND: Annexin V is a human phospholipid binding protein that binds to activated platelets in vitro. We sought to determine the potential of this agent for imaging intracardiac thrombi in swine. METHODS AND RESULTS: Left atrial thrombi were formed by crush injury. In initial nonimaging experiments using intravenous 125I-labeled human annexin V, the mean thrombus/whole blood ratio was 13.4 +/- 4.8 for the entire thrombus using well counting of resected specimens (n = 8). Using intravenously injected 99mTc-labeled human annexin V, the left atrial thrombus/blood ratio by well counting was similar (14.2 +/- 10.6 for the entire thrombus and 26.2 +/- 14.9 for the peak section) (n = 12). The ratio for a control protein, 125I-ovalbumin, was only 1.0 +/- 0.2. 99mTc tomographic imaging was positive (n = 10) or equivocal (n = 2) in all experiments with but negative in 10 controls without left atrial thrombi. By region-of-interest analysis of the tomographic images, the mean left atrial appendage/blood ratio at 2 hours in animals with a thrombus was 3.90 +/- 1.12 compared with 0.84 +/- 0.10 in closed chest controls and 1.01 +/- 0.23 in open chest controls (P < .001). CONCLUSIONS: We conclude that 99mTc-labeled human annexin V detects acute left atrial thrombi in vivo in swine. The combination of a new thrombus detection agent, annexin V, with a 99mTc label may allow in vivo imaging of thrombi in humans.

Animals↗
PubMed1995

Heterogeneity of islet pathology in two infants with recent onset diabetes mellitus.

The mechanisms by which the beta cells of pancreatic islets are destroyed in insulin-dependent diabetes mellitus (IDDM) are poorly understood. In this report the pancreatic histo- and immunopathology of two children, both HLA-DR 3/4, DQ 2/8 positive and who both died from cerebral oedema within a day of clinical diagnosis of IDDM, were investigated. Patient 1, a 14-month-old girl, had a 4-week history of polydipsia and polyuria. Patient 2, a 3-year-old boy, had 2 days of illness. Both patients had a similarly severe loss of insulin cells but differed markedly as to the extent of lymphocytic islet infiltration (insulitis). Apart from insulitis, marked islet macrophage infiltration was demonstrated in both patients with the HAM-56 monoclonal antibody. Neither patient showed aberrant expression of HLA class II antigens on insulin-immunoreactive cells, but allele-specific HLA-DQ8 expression was evident on endothelial cells. Glutamic acid decarboxylase immunoreactivity was detected in both insulin- and glucagon-immunoreactive cells. It is concluded that the heterogeneity of islet pathology, especially insulitis, may reflect different dynamics and extent rather than different pathomechanisms of immune destruction of islets in IDDM.

Child, Preschool↗
PubMed1995

Localization of the plasma membrane Ca(2+)-ATPase isoform PMCA3 in rat cerebellum, choroid plexus and hippocampus.

mRNA encoding rat plasma membrane Ca(2+)-ATPase isoform PMCA3 was localized in the granule cell layer of the cerebellum and in choroid plexus by in situ hybridization with an 35S-labelled oligodeoxynucleotide probe. In order to examine whether this isoform is expressed as a protein in brain, polyclonal antibodies were raised against a peptide corresponding to a C-terminal 18 amino acid sequence of PMCA3 which had been conjugated to bovine serum albumin. Using immunoblot analysis with affinity-purified antibodies, PMCA3 protein was found in rat brain microsomes and cultured neurons. The translated protein had an observed molecular mass of approximately 135 kDa, as predicted from molecular cloning studies. The pattern of localization of PMCA3 in brain using anti-peptide antibodies was consistent with findings from in situ hybridization. PMCA3-like immunoreactive sites were found in the granule cell and molecular layers of rat cerebellum and in choroid plexus, and the pattern of staining suggests that immunoreactive sites are associated with granule cell processes. This conclusion was supported by the finding that growth-associated protein-43, a protein known to be present in axons and nerve terminals, had a pattern of distribution similar to PMCA3 in the molecular layer of cerebellum. Very low levels of PMCA3-like immunoreactivity were associated with Purkinje cell soma or processes, consistent with the low levels of PMCA3 mRNA found in these neurons. PMCA3-like immunoreactivity was lower in hippocampus than in cerebellum; hippocampal CA1 region immunoreactivity was primarily associated with dendritic fields rather than with pyramidal cell bodies. The results demonstrate that a PMCA3-like protein is expressed in neurons of rat brain and is localized primarily in cell processes.

Animals↗
PubMed1995

Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

Animals↗
PubMed1995

Estradiol inhibits the increase of hypothalamic neuropeptide Y messenger ribonucleic acid expression induced by weight loss in ovariectomized rats.

Anorexia and weight loss produced by estradiol (E2) may involve altered expression of neuropeptide Y (NPY) in the hypothalamus. We tested this hypothesis using ovariectomized (OVX) rats by replacing E2 with SILASTIC brand capsule implants and measuring NPY messenger RNA (mRNA) levels in the arcuate nucleus by in situ hybridization. To equalize the effects of weight loss on NPY mRNA expression, E2 deficient OVX rats were pair-fed (n = 10) for 2 days to an OVX group receiving E2 (n = 12). Compared with the weight gain (P < 0.02) of E2 deficient OVX rats (n = 10), OVX rats replaced with E2 and pair-fed OVX rats both had 12.5% lower food intake and weight (P < 0.05). E2 replacement elevated insulin 52% (P < 0.05) and lowered NPY hybridization 32% (P < 0.05) compared with pair-fed controls. During a 2-day fast, E2 replacement (N = 12) attenuated the elevation of NPY mRNA levels 50% (P < 0.01) compared with E2 deficiency (n = 15). Therefore, when E2 is administered to OVX rats, reduced NPY mRNA expression in the hypothalamus is unlikely to be a primary cause of weight loss, although it may contribute to the maintenance of reduced food intake and body weight.

Animals↗
PubMed1995

Effect of intracerebroventricular insulin infusion on diabetic hyperphagia and hypothalamic neuropeptide gene expression.

To test the hypothesis that diabetic hyperphagia results from insulin deficiency in the brain, diabetic rats (streptozotocin-induced) were given an intracerebroventricular (ICV) infusion of saline or insulin (at a dose that did not affect plasma glucose levels) for 6 days. Food and water intake were significantly increased in diabetic rats, but only food intake was affected by ICV insulin. Diabetic hyperphagia was reduced 58% by ICV insulin compared with ICV saline (P < 0.05) and was accompanied by a 69% increase in diabetes-induced weight loss (P < 0.05). To evaluate whether central nervous system (CNS) insulin deficiency affects expression of neuropeptides involved in food intake, in situ hybridization was done for neuropeptide Y (NPY), which stimulates feeding, in the hypothalamic arcuate nucleus and for cholecystokinin (CCK) and corticotropin-releasing hormone (CRH), which inhibit feeding, in the hypothalamic paraventricular nucleus. In diabetic rats, NPY mRNA hybridization increased 280% (P < 0.05), an effect reduced 40% by ICV insulin (P < 0.05). CCK mRNA hybridization increased 50% in diabetic rats (P < 0.05), a response reduced slightly by ICV insulin (P < 0.05), whereas CRH mRNA hybridization decreased 33% in diabetic rats (P < 0.05) and was unchanged by ICV insulin. The results demonstrate that CNS infusion of insulin to diabetic rats reduces both hyperphagia and overexpression of hypothalamic NPY mRNA. This observation supports the hypothesis that a deficiency of insulin in the brain is an important cause of diabetic hyperphagia and that increased hypothalamic NPY gene expression contributes to this phenomenon.

Animals↗
PubMed1995

Autoradiographic assessment of blood flow heterogeneity in the hamster heart.

OBJECTIVE: Provide regional flow measurement in the hearts of small mammals using a new, higher-resolution technique based on the deposition of a molecular marker. METHODS: We determined the instantaneous extraction and retention of the "molecular microsphere" radiolabeled desmethylimipramine in retrogradely perfused hamster hearts. In a separate series of experiments, autoradiography was used to measure regional myocardial deposition densities in hamster hearts of about 0.5 g with spatial area resolution of 16 x 16 microns. RESULTS: Radiolabeled desmethylimipramine is almost 100% extracted during a single transcapillary passage and is retained in the tissue for many minutes. Autoradiographic images demonstrated a spatial flow heterogeneity with standard deviations of 31 +/- 4% of the mean flow (N = 5) in 16 x 16 x 20-micronm3 voxels. This is equivalent to the projections made using fractal relationships from cruder observations obtained with microspheres in the hearts of baboons, sheep, and rabbits. CONCLUSIONS: Autoradiography using a molecular deposition marker provides quantitative information on myocardial flow heterogeneities with resolution at the size of cardiac myocytes. Because the regions resolved are smaller than the volume of regions supplied by single arterioles, the results must slightly exaggerate the true heterogeneity of regional flows.

Animals↗
PubMed1995

Localization of Na, K-ATPase isoforms in the hypothalamus of the rat.

In situ hybridization histochemistry with synthetic oligonucleotide probes was used to localize mRNAs encoding three isoforms of the catalytic (alpha) subunit and one isoform of the (beta) subunit of the Na, K-ATPase in rat hypothalamus using contact film radioautography. 3H-Ouabain binding to specific anatomical nuclei in the hypothalamus was determined using a quantitative radioautographic technique previously developed in our laboratory. Specific hybridization was found with oligonucleotide probes for mRNA encoding alpha 1, alpha 2, alpha 3, beta 1 isoforms of the Na, K-ATPase. High levels of hybridization signal for alpha 3 and beta 1 were found in ventromedial hypothalamus, supraoptic nucleus, paraventricular nucleus and the anterior hypothalamic area. Very low levels of hybridization for all isoforms were found in the optic chiasm. mRNAs encoding alpha 1 and alpha 2 isoforms were expressed at lower levels than alpha 3. The distribution of alpha 2 was consistent with expression in glial cells. Generally, levels of alpha 1 mRNA were higher in the arcuate nucleus than in other hypothalamic regions and very low levels were found in the anterior hypothalamic area. 3H-Ouabain binding was relatively diffuse, consistent with the localization of the synthesized Na, K-ATPase protein in cellular processes. The number of 3H-ouabain binding sites in the paraventricular nucleus was significantly lower than other hypothalamic nuclei studied. The results suggest that Na, K-ATPase isoforms may be differentially expressed in hypothalamic nuclei.

Animals↗
PubMed1994

Weight loss in rats following intraventricular transplants of pancreatic islets.

Because of the body's resistance to permanent weight change, obesity remains a major health problem in modern society. It is hypothesized that the regulatory system defending body weight utilizes pancreatic insulin as an indicator of adiposity to the brain. To take advantage of this negative feedback system, we transplanted neonatal (experiment 1) or adult (experiment 2) pancreatic islets containing insulin-secreting cells into the 3rd ventricle of syngeneic Lewis rats. This resulted in an elevation of the insulin signal within the brain and a significant long-term reduction of body weight. Changes in food intake were consistent with the changes of body weight. The implantation of more islets resulted in a greater reduction of body weight, and changes in weight were inversely correlated with the level of insulin achieved in the cerebrospinal fluid. After the two studies were completed, histological examination revealed the presence of insulin-containing cells within the 3rd ventricle and adjacent hypothalamus. These studies suggest that transplanted insulin-secreting cells may provide a potential therapeutic strategy for maintenance of weight loss.

Animals↗
PubMed1994

Calibration of 14C-plastic standards for quantitative autoradiography with 33P.

Probes labeled with 33P have potential for widespread use in in situ hybridization because they are better able to detect relatively scarce mRNAs compared with probes labeled with 35S, but the relatively short half-life of 33P is a disadvantage when it is used as a radioactivity standard for quantitative autoradiography. To determine if plastic sections containing 14C can be used as standards for quantitative autoradiography with 33P, we co-exposed 33P-labeled liver paste sections and 14C-plastic standards to Hyperfilm beta max. The autoradiographic response of Hyperfilm beta max to these isotopes was almost identical. Second-order polynomial equations obtained from analysis of film relative optical density and radioactivity permitted derivation of tissue-equivalent radioactivity from the film optical densities produced by the 14C standards for 1-14-day exposures. These results validate the use of plastic 14C standards for quantifying 33P used in contact film autoradiography.

Animals↗
PubMed1994

Insulin receptor substrate-1 (IRS-1) expression in rat brain.

IRS-1 is phosphorylated on tyrosine residues after insulin stimulation and participates in the early events of signal transduction in peripheral insulin-sensitive tissues. This study determined whether neuronal populations in the rat olfactory bulb and hippocampus (brain regions which have very high concentrations of insulin receptors) also express IRS-1 and contain phosphotyrosine, using in situ hybridization, receptor binding, and immunocytochemistry. IRS-1 mRNA was colocalized with insulin receptor mRNA in neuron cell bodies of hippocampus and olfactory bulb. Similarly, IRS-1 immunoreactivity in hippocampus and olfactory bulb was concentrated in layers that contain synapses of these neurons and have both high insulin binding and phosphotyrosine levels. Thus, IRS-1 and insulin receptors are coexpressed in discrete populations of neurons, suggesting a signal transduction mechanism by which insulin may influence metabolism and gene expression in the brain.

Animals↗
PubMed1994

Major species variation in the expression of galanin messenger ribonucleic acid in mammalian celiac ganglion.

To determine whether galanin may be a sympathetic neurotransmitter in the pancreas of primates and rats as well as dogs, the expression of the galanin gene was examined in the celiac ganglion of these species by in situ hybridization and RIA. Intense hybridization signal for galanin messenger RNA (mRNA) was observed in every neuronal cell body of the dog celiac ganglion. However, significant hybridization signal for galanin mRNA was seen in only 24 +/- 5% of celiac ganglion cell bodies in monkeys and was absent in rats. RIA of celiac ganglion extracts confirmed this species variation; galanin-like immunoreactivity was highest in dog celiac ganglion (158 +/- 13 pmol/g), present in monkeys (34 +/- 7 pmol/g), and undetectable in rats (< 0.8 pmol/g). In contrast, the celiac ganglia of all three species showed intense hybridization signal for neuropeptide-Y (NPY) mRNA in the majority of neuronal cell bodies (dog, 82 +/- 4%; monkey, 92 +/- 2%; rat, 91 +/- 3%), and the celiac ganglion NPY immunoreactivity content was high in all three species (dog, 1064 +/- 155 pmol/g; monkey, 3180 +/- 745 pmol/g; rat, 3412 +/- 347 pmol/g). Thus, there is a marked species variation in the expression of the galanin, but not the NPY, gene in the celiac ganglion of dogs, monkeys, and rats. These data suggest that galanin is an important sympathetic neurotransmitter in the pancreatic islets of dogs, but not those of primates or rats.

Animals↗
PubMed1994

Pancreatic expression and secretion of human islet amyloid polypeptide in a transgenic mouse.

Islet amyloid polypeptide (IAPP) is a secretory product of the pancreatic beta-cell, which is the primary constituent of the islet amyloid that develops in type II diabetes. To study the role the inherent amyloidogenicity of human IAPP (hIAPP) plays in the formation of islet amyloid deposits and to investigate a possible hormonal role for IAPP, transgenic mice expressing hIAPP were developed. The transgene was composed of a fragment of an hIAPP cDNA linked to the rat insulin II promoter. One line of transgenic mice expressed the transgene and synthesized hIAPP in their pancreatic islets. IAPP-like immunoreactivity in pancreatic extracts and plasma were two- to threefold greater in the transgenic mice compared with nontransgenic control mice. Although plasma concentrations of immunoreactive insulin (IRI) and glucose were equal in transgenic and control mice, the pancreatic content of IRI was nearly twofold greater in the transgenic animals, and proinsulin mRNA was significantly elevated, suggesting increased rates of insulin biosynthesis. Pancreatic samples obtained from transgenic mice up to 19 months of age had no evidence of islet amyloid. These results indicate that an increased level of synthesis of the amyloidogenic hIAPP is not sufficient to cause islet amyloid deposition. However, the increased synthesis and storage of insulin in the islets of the transgenic mice are consistent with either a direct regulatory effect of IAPP on the beta-cell or indirect stimulation of insulin production through IAPP-induced insulin resistance.

Amyloid↗
PubMed1993

Immunocytochemical detection of insulin receptor substrate-1 (IRS-1) in rat brain: colocalization with phosphotyrosine.

In peripheral insulin-sensitive tissues, insulin receptor substrate (IRS-1) undergoes tyrosine phosphorylation immediately after cells are stimulated by insulin or insulin-like growth factor-1 (IGF-1), and may function as a molecular link between insulin/IGF-1 receptor tyrosine kinases and enzymes regulating cell growth and metabolism. A fundamental question pertaining to insulin/IGF-1 action in the brain is whether IRS-1 is expressed by neurons. In this study, the distribution of cells containing immunoreactivity to IRS-1 in the brain was determined by immunocytochemistry with polyclonal IRS-1 antiserum, and compared to the localization of immunostaining for phosphotyrosine using polyclonal phosphotyrosine antiserum. The immunostaining results with ABC-peroxidase method and cryostat sections showed the presence of IRS-1 immunoreactivity in many neuron cell bodies throughout the rat forebrain, particularly in the habenula, cerebral cortex and piriform cortex. In the hypothalamus, IRS-1 immunostaining was present in neurons of the paraventricular nucleus, supraoptic nucleus, and arcuate nucleus. The choroid plexus stained intensely for IRS-1. The populations of cells that stained for IRS-1 also showed strong immunostaining for phosphotyrosine. Studies at the cellular level are needed to verify coexpression of IRS-1 and receptors for insulin or IGF-1 by the same neurons, as well as in cells of the choroid plexus. The present results are the first demonstration of IRS-1 expression by neurons in adult mammalian brain. These findings are consistent with the hypothesis that insulin and IGF-1 actions in the brain involve signal transduction mechanisms common to those found in peripheral tissues.

Animals↗
PubMed1993

Food intake and estradiol effects on insulin binding in brain and liver.

Three groups of ovariectomized rats were treated for 6 days: 1) estradiol benzoate (100 micrograms/kg) (SC) and fed ad lib; 2) vehicle-injected controls fed the same amount of food as eaten by estradiol-treated rats; 3) vehicle-injected, free-feeding controls. Specific binding of insulin to liver and hypothalamus slices was measured by quantitative film autoradiography. Estradiol-treated rats lost weight (p < 0.001) and had elevated plasma insulin (p < 0.01). Liver insulin binding in rats with estradiol treatment was greater (p < 0.01) than in rats without estradiol, but was less (p < 0.05) than in controls fed the same food levels as consumed by the estradiol-treated rats. Therefore, with equal food intake, estradiol decreased liver insulin binding. Insulin binding in the dorsomedial, ventromedial, and arcuate nuclei of the hypothalamus was unchanged by food intake or estradiol, however. Thus, altered insulin binding in the arcuate, ventromedial, or dorsomedial nuclei of the hypothalamus is probably not involved in the effects of insulin or estradiol on food intake.

Animals↗
PubMed1993

NPY and galanin in a hibernator: hypothalamic gene expression and effects on feeding.

Neuropeptides such as neuropeptide Y (NPY) and galanin may play a role in regulating the pronounced seasonal changes in food intake shown by golden-mantled ground squirrels (Spermophilus saturatus). We used in situ hybridization histochemistry to localize the expression of NPY and galanin mRNA in the hypothalamus of normally feeding animals. NPY mRNA was abundantly expressed in the arcuate nucleus, while galanin mRNA was concentrated in both the arcuate nucleus and the dorsomedial nuclei. When NPY (0.1, 0.5, 2, and 8 micrograms) or galanin (0.1, 0.5, 2, and 8 micrograms) were injected into the third cerebral ventricle, food intake was significantly and dose-dependently increased over the subsequent 30 min. NPY stimulated significant increases in food intake for up to 2 h whereas galanin's effect did not extend beyond 30 min. Our results suggest that hibernating and nonhibernating rodents share common neural substrates for the regulation of food intake. Seasonal modulation of these neural pathways may contribute to annual cycles of food intake in hibernating mammals.

Analysis of Variance↗