Urocortin 3 transgenic mice exhibit a metabolically favourable phenotype resisting obesity and hyperglycaemia on a high-fat diet.

AIMS/HYPOTHESIS: Urocortins are the endogenous ligands for the corticotropin-releasing factor receptor type 2 (CRFR2), which is implicated in regulating energy balance and/or glucose metabolism. We determined the effects of chronic CRFR2 activation on metabolism in vivo, by generating and phenotyping transgenic mice overproducing the specific CRFR2 ligand urocortin 3. METHODS: Body composition, glucose metabolism, insulin sensitivity, energy efficiency and expression of key metabolic genes were assessed in adult male urocortin 3 transgenic mice (Ucn3(+)) under control conditions and following an obesogenic high-fat diet (HFD) challenge. RESULTS: Ucn3(+) mice had increased skeletal muscle mass with myocyte hypertrophy. Accelerated peripheral glucose disposal, increased respiratory exchange ratio and hypoglycaemia on fasting demonstrated increased carbohydrate metabolism. Insulin tolerance and indices of insulin-stimulated signalling were unchanged, indicating these effects were not mediated by increased insulin sensitivity. Expression of the transgene in Crfr2 (also known as Crhr2)-null mice negated key aspects of the Ucn3(+) phenotype. Ucn3(+) mice were protected from the HFD-induced hyperglycaemia and increased adiposity seen in control mice despite consuming more energy. Expression of uncoupling proteins 2 and 3 was higher in Ucn3(+) muscle, suggesting increased catabolic processes. IGF-1 abundance was upregulated in Ucn3(+) muscle, providing a potential paracrine mechanism in which urocortin 3 acts upon CRFR2 to link the altered metabolism and muscular hypertrophy observed. CONCLUSIONS/INTERPRETATION: Urocortin 3 acting on CRFR2 in skeletal muscle of Ucn3(+) mice results in a novel metabolically favourable phenotype, with lean body composition and protection against diet-induced obesity and hyperglycaemia. Urocortins and CRFR2 may be of interest as potential therapeutic targets for obesity.

Glucocorticoids differentially regulate the expression of CRFR1 and CRFR2α in MIN6 insulinoma cells and rodent islets.

Urocortin 3 (Ucn 3), member of the corticotropin-releasing factor (CRF) family of peptide hormones, is released from ß-cells to potentiate insulin secretion. Ucn 3 activates the CRF type-2 receptor (CRFR2) but does not activate the type-1 receptor (CRFR1), which was recently demonstrated on ß-cells. While the direct actions of Ucn 3 on insulin secretion suggest the presence of cognate receptors within the islet microenvironment, this has not been established. Here we demonstrate that CRFR2α is expressed by MIN6 insulinoma cells and by primary mouse and human islets, with no detectable expression of CRFR2ß. Furthermore, stimulation of MIN6 cells or primary mouse islets in vitro or in vivo with glucocorticoids (GCs) robustly and dose-dependently increases the expression of CRFR2α, while simultaneously inhibiting the expression of CRFR1 and incretin receptors. Luciferase reporters driven by the mouse CRFR1 or CRFR2α promoter in MIN6 cells confirm these differential effects of GCs. In contrast, GCs inhibit CRFR2α promoter activity in HEK293 cells and inhibit the expression of CRFR2ß in A7r5 rat aortic smooth muscle cells and differentiated C2C12 myotubes. These findings suggest that the GC-mediated increase of CRFR2α depends on the cellular context of the islet and deviates from the GC-mediated suppression of CRFR1 and incretin receptors. Furthermore, GC-induced increases in CRFR2α expression coincide with increased Ucn 3-dependent activation of cAMP and MAPK pathways. We postulate that differential effect of GCs on the expression of CRFR1 and CRFR2α in the endocrine pancreas represent a mechanism to shift sensitivity from CRFR1 to CRFR2 ligands.

Effects of feeding time on nitrogen capture by lactating dairy cows grazing rye pasture.

The objective of this experiment was to determine whether varying times at which a partial mixed ration was fed, either before or after grazing, affected N utilization from rye pasture and thus affected milk yield and components. Sixteen Holstein cows were fed a partial mixed ration (PMR) either at 0700, 0830, or 1100 h. Cows were milked at 0900 h and turned out to graze at 0930 h. Treatments represented feeding times 2.5 h and 1 h before grazing and immediately after grazing. The study was conducted as a 3 x 3 Latin square with three 17-d periods. There were no significant differences among treatments for pasture intake or yield of milk or milk components. Milk yield, fat %, and protein % were 29.4, 29.6, and 29.3 kg, 3.5, 3.5, and 3.4%, and 3.4, 3.5, and 3.4% for treatments, respectively. The milk urea levels were 15.6, 15.1, and 15.5 mg/dl, and were not different among treatments. Blood samples were collected on the last day of each period at 0645, 0845, 1045, 1200, and 1400 h. Blood urea nitrogen (BUN) was measured as an indicator of ruminal N capture. Concentrations were not significantly different among diets before grazing; however, they were significantly different among all treatments approximately 1 h after cows were removed from pasture. Cows fed at 0700 h, 2 h before grazing, maintained lower BUN levels across the 7 h during which the blood samples were collected. Cows that ate the PMR immediately after grazing maintained the highest BUN. Feeding a PMR to cows that graze at different times before and after grazing affected the capture of ruminal N, as indicated by differences in the levels of BUN, but there was no effect on yield of milk or milk components.

Activin and inhibin binding to the soluble extracellular domain of activin receptor II.

Activins and inhibins belong to the transforming growth factor-beta-like superfamily of growth and differentiation factors that exert pleiotropic effects in many target tissues. Heteromeric association of activin with two structurally related receptor serine/threonine kinases, activin receptor types I and II, initiates downstream signaling events. The extracellular domain of type II mouse activin receptor (ActRII ECD) was expressed in the baculovirus system, purified in three steps by lectin affinity, anion exchange, and reverse phase chromatography, and further characterized by mass spectrometry. The reduction in the apparent size of the purified ActRII ECD on SDS-PAGE after treatment with glycosidases provided evidence for N- and O-linked oligosaccharides. Specific receptor/ligand complexes of [125I] activin A to ActRII ECD or [125I]ActRII ECD to activin A were analyzed by cross-linking and immunoprecipitation. Two major radiolabeled bands were observed on SDS-PAGE with mobilities consistent with the expected size of ActRII ECD/betaA or ActRII ECD/betaAbetaA. When inhibin A was cross-linked to [125I]ActRII ECD, a slower migrating complex corresponding to ActRII ECD/betaAalpha was also observed. The apparent dissociation constant (Kd) for activin A binding to ActRII ECD was 2-7 nM. This Kd value is approximately an order of magnitude greater than that of the full-length membrane-associated type II receptor. Treatment of cultured rat anterior pituitary cells with ActRII ECD attenuated FSH secretion in response to exogenous activin A or endogenous activin B. These data indicate that the soluble ActRII ECD has structural determinants that are sufficient for high affinity ligand binding.

Cloning and characterization of human urocortin.

Urocortin, a new member of the CRF peptide family which also includes urotensin I and sauvagine, was recently cloned from the rat midbrain. The synthetic replicate of urocortin was found to bind with high affinity to type 1 and type 2 CRF receptors and, based upon its anatomic localization within the brain, was proposed to be a natural ligand for the type 2 CRF receptors. Using a genomic library, we have cloned the human counterpart of rat urocortin and localized it to human chromosome 2. Human and rat urocortin share 95% identity within the mature peptide region. Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells. In addition, the CRF-binding protein binds human urocortin with high affinity and can prevent urocortin-stimulated ACTH secretion in vitro. The inhibitory effect of the CRF-binding protein on human urocortin can be blocked by biologically inactive CRF fragments, such as CRF(9-33).

Polarization states and output powers of a CO(2) laser with an electro-optic phase retarder.

We study the power output and polarization state of a CO(2) laser that contains both passive polarization-sensitive elements (Brewster plates) and an active phase-retarding electro-optic modulator (CdTe crystal). The theoretical threshold condition (i.e., the retardation required to extinguish the laser) is obtained from a cavity round-trip Jones matrix. We show that adding extra Brewster plates may be counterproductive in that the required retardation voltage increases. We calculated the output power, which depends on the saturated gain properties of the active media, by iterative modeling, with the elliptically polarized light propagated repeatedly around the cavity. We compare the theory with results from a folded rf-excited waveguide CO(2) laser (Laser Ecosse Model CM3500). Theory and experiment are in fair agreement for low powers. As expected, at high powers there are extra difficulties in modeling the cavity behavior, probably because of distortions induced in and by the crystal.

Inactivation of activin-dependent transcription by kinase-deficient activin receptors.

Activin, a member of the transforming growth factor-beta superfamily, binds to two classes of cell surface receptors. These receptors, designated type I and type II, are structurally related members of transmembrane serine kinase superfamily. Antibodies specific for either type I or type II activin receptor can coprecipitate complexes containing both affinity-labeled receptors from activin-responsive cells. Two type I receptors show cell-specific expression and associate with the ligand-binding, type II receptors. To investigate the roles of the cytoplasmic receptor domains in signaling through a heteromeric ligand receptor complex, we have made kinase-deficient activin receptors and correlated their losses in kinase activity with inhibitory effects on an activin-dependent transcriptional response in activin-responsive cell lines. Wild-type activin type II receptors phosphorylate activin type I receptors in transfected COS cells. In contrast, kinase-deficient activin type II receptors fail to phosphorylate type I receptors in transfected COS cells and act as dominant negative mutants to block activin-induced transcriptional activity in both Chinese hamster ovary and K562 (human erythroleukemia) cells. Kinase-deficient activin type IB receptors also block activin-induced transcriptional activity in both Chinese hamster ovary and K562 cells, whereas kinase-deficient activin type I receptors have no effect in either cell line. These results indicate that kinase activities of both type II and type I receptors are required for activin signaling, and that the two type I receptors, which are expressed in a tissue-specific manner, are functionally distinct.

Monomeric activin A retains high receptor binding affinity but exhibits low biological activity.

Activins are multipotent hormones/growth factors that belong to the transforming growth factor-beta (TGF-beta) superfamily. Like TGF-beta s, activins have 9 conserved cysteine residues and are disulfide-bonded dimers. Based on the three-dimensional structure of TGF-beta 2, we deduced Cys80 in activin A to form the intermolecular disulfide bond. To obtain a monomeric form of activin, Cys80 was exchanged for a serine residue by polymerase chain reaction mutagenesis. The mutant protein was expressed in a baculovirus/insect cell expression system. The molecular mass of this mutant activin was determined to be 13 kDa (consistent with a single chain form of the protein) by SDS-polyacrylamide gel electrophoresis and by laser desorption mass spectroscopy. When this mutant monomeric activin was incubated with cells that expressed either the activin type IIB receptor or both the type I and type IIB receptors, its affinity was found to be 20% of that of native activin on a mass basis. Binding affinity determined using the mouse pituitary cell line AtT 20 was 10% of that of native activin A. Biological potency, however, as determined by the mutant protein's ability to release FSH from anterior pituitary cells in primary culture and by its ability to suppress basal ACTH secretion form AtT 20 cells, was only 1% of that of the native protein. This discrepancy of an order of magnitude between binding and biological activity is consistent with a model in which dimerization of the hormone is not necessary for high affinity binding to its receptor(s) while being essential for efficient signal transduction.

Enhancement of coherent laser radar performance by predetection amplification.

A long-path carbon dioxide laser amplifier, based on a coaxial radio frequency discharge gain medium combined with a Herriott cell folding system, is investigated as a predetection amplifier in a coherent laser radar system. The amplifier signal gain and noise characteristics are measured, and the signal-to-noise enhancement produced in a Doppler radar system is determined over a range of operating conditions of the amplifier. A maximum enhancement factor of 12.4 dB is measured for a cw gain cell, which is in good agreement with the theoretical prediction.

Higher moments of scattered light fields by heterodyne analysis.

In heterodyne detection (such as in coherent lidar) the optical local oscillator defines a single mode of the incoming-signal light field; this single-mode selectivity has been previously predicted to preserve the full fluctuation character of scattered light. This is in contrast with direct-detection schemes, as in photon-correlation spectroscopy, where aperture averaging usually reduces the range of fluctuations. Examples of Gaussian and non-Gaussian statistics in laser light scattered from a moving ground-glass screen have been studied. This simple laboratory experiment has several advantages over equivalent direct-detection schemes and has been shown to yield experimentally the theoretically predicted factorial intensity moments (up to the seventh order) that result from zero-mean, circulo-complex Gaussian statistics.

Characterization and the regulation of inhibin/activin subunit proteins of cultured rat anterior pituitary cells.

The production of inhibin/activin by cultured rat anterior pituitary cells was evaluated using specific antisera to inhibin/activin alpha, beta A, and beta B subunit proteins (anti-alpha, anti-beta A, and anti-beta B). Cellular or secreted proteins recognized by the antisera were immunoprecipitated from metabolically labeled cells then analyzed by denaturing polyacrylamide gel electrophoresis. Immunoreactive inhibin/activin beta B proteins were visualized in both cell lysates and the media. Experiments with anti-beta B confirmed that activin-B (beta B beta B) is a local secretory product of cultured rat anterior pituitary cells. The secreted beta B-immunoreactive protein band had an apparent size of 24-25 kilodaltons (kDa) or 14-15 kDa, consistent with the size of unreduced beta B dimer or reduced monomer, respectively. Cell lysates contained two proteins that were specifically immunoprecipitated by anti-beta B. One of these had a mobility of greater than 95 kDa (unreduced) or 55-60 kDa (reduced), probably representing dimers or monomers of the beta B precursor, respectively. The second 14- to 15-kDa (reduced and unreduced) immunoreactive beta B protein band was verified to be the mature beta B monomer. Mature heterodimeric inhibin-B (alpha beta B) was not detected by either anti-alpha or anti-beta B. Multiple protein species, however, were observed to be specifically immunoprecipitated by incubation of cell lysates with anti-alpha. Mature beta A monomer was not detected in any of the samples. The regulation of cellular beta B production was monitored by evaluating its rate of synthesis in pulse-labeled cells. Treatment with either forskolin or 12-O-tetradecanoylphorbol acetate enhanced the rate of [35S]cysteine incorporation into the cellular 14- to 15-kDa beta B monomer, indicating that the activation of either protein kinase A or protein kinase C regulates its production. The rate of cellular beta B accumulation was also regulated by activin-A, inhibin-A, and follistatin; activin-A caused a 30% inhibition in contrast to the 70% stimulation by treatment with either inhibin-A or follistatin. Equimolar concentrations of activin-A and follistatin prevented the net effect produced by either factor alone. None of the immunoreactive alpha-forms was detectable under similar pulse-labeling conditions, and there was no apparent change in their level after labeling to equilibrium (up to 48 h). The observed changes in beta B accumulation may, therefore, reflect the regulated production of pituitary activin-B. Taken together, these results suggest that locally produced activin-B or gonadal activins exert an inhibitory tone on the production of pituitary activin-B and that this negative-feedback control is in turn modulated by inhibins and follistatins. The relative importance of pituitary and gonadal activins, inhibins, and follistatins in the proposed regulatory loop remains to be established.

Activin-A regulates follistatin secretion from cultured rat anterior pituitary cells.

The activin-binding protein, follistatin (FS), was immunoprecipitated from metabolically labeled rat anterior pituitary cells or their media using a specific antiserum to purified porcine FS (anti-FS). Several immunoreactive proteins, including one that had a mobility in the range of 42-44 kilodaltons (kDa), were detected in the cell lysates. When immunoprecipitates of the culture medium were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, a broad 35- to 46-kDa or 39- to 53-kDa band was visualized under unreducing or reducing conditions, respectively. Upon deglycosylation by treatment with N-glycosidase-F, the secreted product migrated as a sharp protein band with an apparent size of 35 kDa. The identity or the relatedness of the immunoprecipitated proteins to FS was verified by the ability of the C-terminally truncated form of recombinant human FS (rhFS288) to compete for binding to anti-FS. When the cultured rat anterior pituitary cells were treated with either forskolin or 12-O-tetradecanoylphorbol acetate, the accumulation of FS in the culture medium was stimulated by approximately 2.5-fold. These observations suggest that the activation of either the protein kinase A or the protein kinase C signaling pathway has a stimulatory effect on anterior pituitary FS production. A more dramatic stimulation of FS secretion (up to 7-fold) was observed when the rat anterior pituitary cells were treated with activin-A. The concentration dependence for this effect was within the same range that has been reported for most of the actions of activin-A. Inhibin-A suppressed basal FS secretion and blocked its stimulation by activin-A. To determine if locally produced FS exerts an influence on the response of gonadotropes to activins, the effects of anti-FS on FSH secretion were monitored. The ability of this FS antiserum to immunoneutralize the activity of FS was initially confirmed; anti-FS attenuated the inhibitory action of exogenous follistatin on FSH secretion. Treatment of cells with the antiserum increased the apparent sensitivity of gonadotropes to submaximal concentrations of activin-A. Moreover, the presence of the antiserum lowered the concentration of activin-A that was required to produce the maximum amount of FSH secretion, without changing the magnitude of the response. These results suggested that locally produced FS interferes with the secretory response of gonadotropes to activins. Changes in locally secreted FS may, therefore, represent a mechanism by which the response of rat anterior pituitary cells to incoming stimuli are tightly regulated.

Alpha 2-macroglobulin is a binding protein of inhibin and activin.

The development of a two-site immunoassay for dimeric inhibin resulted in the detection of higher mol wt substances in follicular fluid that interfere with the measurement of dimeric inhibin by this assay, but not by a previously characterized inhibin alpha-subunit RIA. Therefore, we initiated a study to identify inhibin- and activin-binding proteins in biological fluids using 125I-labeled recombinant human inhibin-A and activin-A. By gel permeation chromatography, we found that [125I] inhibin A or [125I]activin-A incubated with either plasma or follicular fluid became associated with several higher mol wt proteins. The lowest mol wt complex eluted with a retention time similar to that of [125I] inhibin or [125I]activin bound to porcine follistatin, a known inhibin- and activin-binding protein. The largest mol wt complex eluted near the void volume, coincident with human alpha 2-macroglobulin (h alpha 2 M) incubated with [125I]inhibin or [125I]activin. Human plasma was incubated with [125I]inhibin or [125I]activin, and putative binding proteins were chemically cross-linked and analyzed by polyacrylamide gel electrophoresis. Cross-linked 125I-labeled inhibin or activin plasma binding protein complexes comigrated with [125I] inhibin or [125I]activin cross-linked to h alpha 2M. Antiserum to h alpha 2M precipitated 125I-labeled inhibin- and activin-binding proteins in human plasma that migrated identically in polyacrylamide gel electrophoresis to complexes formed between [125I]inhibin or [125I]activin cross-linked to h alpha 2M. Excess unlabeled inhibin or activin decreased the labeling of h alpha 2M and high mol wt human plasma binding proteins by [125I]inhibin or [125I]activin. These results demonstrate that in addition to follistatin, alpha 2M is a binding protein of both inhibin and activin. Although the physiological relevance of the inhibin-alpha 2M and activin-alpha 2M interaction is unknown, alpha 2M may play a role in the delivery or clearance of inhibin and activin similar to that proposed for transforming growth factor-beta and a number of other growth factors.

Characterization and regulation of corticotropin-releasing factor in the human hepatoma NPLC-KC cell line.

Corticotropin-releasing factor (CRF) has a broad distribution throughout the brain and periphery and has been shown to be present in certain human tumors. We have identified a human hepatoma cell line, NPLC-KC, that contains and secretes immunoreactive CRF (irCRF). Cyclohexyl-silica extracted supernatant from these cells displaced iodinated human CRF (hCRF) in our CRF radioimmunoassay in a manner parallel to the hCRF standard, stimulated release of ACTH from cultured primary rat anterior pituitary cells in a dose-responsive manner. In addition, multiple bioactive and immunoreactive forms of CRF were secreted from these cells as determined by gel permeation chromatography. To determine if irCRF secretion and synthesis could be modulated in the NPLC-KC cells, we tested the ability of factors and hormones to regulate CRF release from and synthesis in these cells. Phorbol 12-myristate 13-acetate (PMA) and forskolin stimulated irCRF release and synthesis, implicating calcium/phospholipid- and cAMP-dependent protein kinases in the regulation of these cells. The cytokine, interleukin-1, was a potent secretagogue of irCRF and this was effect-additive with the increase observed with forskolin, but not with PMA. The glucocorticoid dexamethasone exhibited negative regulation of both basal and stimulated secretion and synthesis of irCRF from the NPLC-KC cell line. Thus, the control of irCRF production by the NPLC-KC human hepatoma cell line exhibits distinct similarities to regulation of CRF in the rat hypothalamus. This cell line should therefore be useful for studying regulation of the synthesis, processing and secretion of mammalian CRF in vitro.

Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.

Characterization of melanin-concentrating hormone from rat hypothalamus.

A melanin-concentrating hormone (MCH)-like peptide was isolated from rat hypothalamus by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against salmon MCH, and two steps of HPLC using octadecyl columns. Several zones of immunoreactivity were isolated, and Edman degradation in a gas phase sequencer indicated that the amino acid sequence of all zones was identical. Rat hypothalamic MCH is a nonadecapeptide which differs from salmon MCH by an N-terminal extension of two amino acids and four additional substitutions. Rat MCH has the following primary structure: Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Gln- Val.

Observation of Brillouin scattering from single muscle fibres.

The propagation of sound waves along relaxed single fibres of glycerinated rabbit psoas muscle has been observed using Brillouin scattering at frequencies up to 1.6 GHz. Two types of waves were observed: one with a velocity of 1508 +/- 7 m s-1, which is attributed to sound waves in intra-cellular saline, the other with a velocity of 912 +/- 25 m s-1, which is attributed to waves propagating along the protein filaments within individual sarcomeres. The latter sound velocity is much higher than that which has been reported by Stienen and Blangé (1985) for 50 microseconds tension transients, and the difference is attributed to the much higher stiffness of the protein filaments compared to the cross-bridges which determine the low-frequency elasticity of muscle fibres.

Evidence of a tropospheric aerosol backscatter background mode.

Climatologies of aerosol backscatter profiles, derived from airborne a nd ground-basedC 02 lidarr eturns,i ndicate a high frequency of occurrence for a narrow range of low backscatter values within the troposphere-a background mode.