Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.

To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.

SOCS-3 expression in leptin-sensitive neurons of the hypothalamus of fed and fasted rats.

Treatment of rodents with exogenous leptin increases SOCS-3 mRNA levels in the arcuate nucleus (ARC) and dorsomedial nucleus (DMN) of the hypothalamus. To determine if SOCS-3 gene activity in the hypothalamus could be influenced by changes in physiological levels of circulating leptin, we performed in situ hybridization (ISH) and immunostaining for SOCS-3 expression in fed vs. fasted (48 h) rats. The ARC and DMN were the only regions of the diencephalon that showed SOCS-3 ISH and the autoradiographic ISH signal for SOCS-3 mRNA was visibly less in the ARC and DMN of fasted rats. The ISH signal for SOCS-3 mRNA was decreased 70% in the ARC and 90% in the DMN (to background levels) when animals were fasted (P<0.01), consistent with decreased immunostaining for SOCS-3 protein observed in the fasted rats. Double fluorescence ISH (FISH) analyses showed colocalization of SOCS-3 mRNA with mRNAs for NPY and POMC in the ARC. These findings are consistent with increased leptin signaling to the NPY and POMC neurons in the ARC by physiological levels of circulating leptin during normal feeding. Therefore, changes in SOCS-3 mRNA levels in the ARC and DMN can be viewed as an indicator of relative physiological leptin signaling to the hypothalamus and also identify cells responding directly to leptin signaling through its cognate receptor.

Leptin binding in the arcuate nucleus is increased during fasting.

The arcuate nucleus (ARC) mediates the anorexic effects of leptin and expresses the long form (Ob-Rb) of the leptin receptor. To determine whether ARC leptin binding increases when plasma leptin levels are low during fasting, [125I]-leptin specific binding to rat brain slices was measured by quantitative autoradiography. [125I]-leptin specific binding was dense in the ARC and increased 2-fold after a 48-h fast (P<0.001). These findings suggest that leptin receptor binding in the ARC is upregulated during fasting and that fasting changes the sensitivity of the ARC to leptin.

Regulation of the megakaryocytic glycoprotein IX promoter by the oncogenic Ets transcription factor Fli-1.

Glycoprotein (GP) IX is a subunit of the von Willebrand receptor, GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced promoter activity. However, the Ets protein(s) that regulated GPIX promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T kidney fibroblasts with a Fli-1 expression vector shows that the oncogenic protein Fli-1 can transactivate the GPIX promoter when an intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells. Comparative studies showed that Fli-1 was also able to transactivate the GPIbalpha and, to a lesser extent, the GPIIb promoter. Immunoblot analysis identified Fli-1 protein in lysates derived from platelets. In addition, expression of Fli-1 was identified immunohistochemically in megakaryocytes derived from CD34(+) cells treated with the megakaryocyte differentiation and proliferation factor, thrombopoietin. These results suggest that Fli-1 is likely to regulate lineage-specific genes during megakaryocytopoiesis.

Leptin receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus.

The decline of leptin (Ob protein) concentrations during fasting is implicated as a signal for increasing the expression of the orexigenic peptide neuropeptide Y (NPY) in the hypothalamus. To test the hypothesis that the effects of food intake on arcuate nucleus NPY activation are mediated by leptin, we performed simultaneous triple in situ hybridization colocalization studies to determine whether the subset of NPY neurons that are activated by fasting preferentially expresses the long form of the leptin receptor (Ob-Rb). Thus, mRNAs encoding NPY and pro-opiomelanocortin (POMC) were colocalized in the arcuate nucleus of fed and fasted rats by fluorescence in situ hybridization in combination with isotopic in situ hybridization for Ob-Rb mRNA. In fed animals, 47% of arcuate nucleus neurons containing NPY mRNA also contained Ob-Rb mRNA, compared with 79% of POMC neurons (P < 0.01). After a 2-day fast, the number of arcuate nucleus neurons with NPY mRNA increased 50% (P < 0.05); the number of these that coexpressed Ob-Rb increased twofold (P = 0.013). Furthermore, Ob-Rb mRNA hybridization in individual NPY neurons increased by 64% (P < 0.02). In contrast, the number of POMC neurons that coexpressed Ob-Rb was unchanged. A significant interpretation of these findings is that the NPY neurons that do not express detectable levels of Ob-Rb mRNA are not activated by fasting, whereas the NPY neurons that are activated by fasting are the ones that express Ob-Rb. These data demonstrate a significant physiological difference between NPY neurons that express Ob-Rb and those that do not. The results support the conclusion that the effect of food intake on NPY neurons is mediated by the direct action of leptin via Ob-Rb receptors expressed by these NPY cells. The results also indicate that expression of Ob-Rb is a defining phenotypic characteristic of the subset of arcuate nucleus NPY neurons that are activated by fasting and play a central role in the adaptive response to negative energy balance.

Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus.

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Neuropeptide Y-like gene expression in the salmon brain increases with fasting.

In mammals there is a well-established connection between neuropeptide Y (NPY) and the balance between energy intake and expenditure: NPY stimulates food intake and the hypothalamus shows a dramatic increase in NPY mRNA in response to fasting. The widespread occurrence of NPY in the brains of all vertebrates investigated raises the possibility that NPY may be involved in food intake and energy balance regulation in nonmammalian vertebrates as well. We used in situ hybridization to examine whether brain NPY-like gene expression is involved in energy balance regulation in salmon. A radiolabeled oligonucleotide probe was employed to screen the salmon forebrain and parts of the midbrain for NPY-like mRNA. Distribution of NPY-like gene expression was determined, followed by examination of brains from fed or food-deprived chinook and coho salmon. Regions expressing NPY-like mRNA were the caudoventral telencephalon, preoptic area, thalamus, optic tectum, and caudal hypothalamus. The region showing a difference in NPY-like gene expression between fed and fasted individuals was the preoptic area of the hypothalamus where significantly greater hybridization signal area was found with fasting. Plasma insulin levels were also shown to differ, with fasted animals having significantly lower insulin levels. These results suggest that the role of NPY-like peptides in the regulation of energy balance may have arisen early in vertebrate evolution.

An islet-cell protein tyrosine phosphatase is a likely precursor to the 37-kDa autoantigen in type 1 diabetes: human and macaque sequences, tissue distribution, unique and shared epitopes, and predictive autoantibodies.

BACKGROUND: We sought to identify novel islet-cell autoantigens to better understand the pathogenesis, prediction, and immunotherapy of type 1 diabetes. MATERIALS AND METHODS: Macaque and human islet cDNA libraries expressed in mammalian cells were screened with human diabetes sera. A positive clone was sequenced directly and after 5' rapid amplification of cDNA ends (RACE). Northern blotting and in situ hybridization revealed the tissue distribution of the corresponding protein. Antigen, expressed by in vitro translation, and tryptic peptides were analyzed by SDS-PAGE. For the immunoprecipitations, 183 diabetic, 60 prediabetic, and 91 control sera were used. Truncated antigens were used in immunoprecipitations for epitope mapping. Recombinant antigen expressed in transfected fibroblasts was used in competition assays. RESULTS: Sequencing yielded an 111-kDa, 1,013 amino acid, transmembrane protein (M1851) containing consensus protein tyrosine phosphatase (PTPase) sequence. M1851 was 77% identical in the intracellular domain, but only 31% identical extracellularly, to the islet-cell autoantigen ICA512. mRNA localized to brain, prostate, pancreatic islets, and adrenal medulla. After limited trypsinization, the in vitro translated antigen was 37 kDa. M1851 was recognized by 47% of prediabetes sera, 31% of new diabetes sera, but only 1% of healthy controls. Only 1/73 sera binding M1851 failed to bind ICA512, whereas 42/114 binding ICA512 did not bind M1851. M1851 reactivity was not fully displaced by ICA512 in 24/49 sera. Removing the C-terminal 27, 80, or 160 amino acids of M1851 decreased reactivity by 70%, 90%, and 100%, respectively. CONCLUSIONS: This new islet-cell PTPase is likely to be the precursor to the 37-kDa tryptic fragment antigen. It is structurally related to ICA512 but has distinct diabetes autoantibody epitopes located at the C terminus.

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.

Plasma membrane Ca(2+)-ATPase isoforms: distribution of mRNAs in rat brain by in situ hybridization.

Several mRNAs which encode for isoforms of the plasma membrane Ca(2+)-transport ATPase (PMCA) are present in adult rat brain. Using in situ hybridization with antisense oligonucleotide probes we found complex patterns of specific hybridization for three isoforms (PMCA1-3). Each rat brain region studied exhibited a distinct pattern of expression of isoforms. PMCA1 mRNA, which is widely distributed in rat tissues, was highest in CA1 pyramidal cells of hippocampus and very low in hypothalamic nuclei, cerebellum and choroid plexus. PMCA2 mRNA was highest in Purkinje cells of cerebellum and low in caudate-putamen, hypothalamic nuclei, habenula and choroid plexus. The highest levels of PMCA3 mRNA were found in habenula and choroid plexus. The PMCA1-3 isoforms appeared to be expressed primarily in neurons since hybridization was detected neither in white matter nor in regions rich in astrocytes. In different regions, different levels of expression of each PMCA mRNA may underlie specialized requirements for calcium homeostasis in specific neurons.

Over 95 per cent sensitization against allogeneic leukocytes following single massive blood transfusion.

The development and persistence of leukoagglutinating and lymphocytotoxic alloantibodies were systematically studied in 54 patients who had received single massive blood transfusions because of open-heart surgery. Of these patients over 95% were sensitized when tested for both leukoagglutinins and lymphocytotoxins, whereas 74% were found to be immunized when searched for cytotoxic antibodies only. The optimal time for detecting antibodies was 2 weeks after blood transfusions.