Circadian Neuroendocrine Role in Age-Related Changes in Body Fat Stores and Insulin Sensitivity of the Male Sprague-Dawley Rat

A role for circadian neuroendocrine rhythms in the age-related development of obesity and insulin resistance was investigated in the male Sprague-Dawley rat. The phases and amplitudes of the plasma rhythms of several metabolic hormones (i.e. corticosterone, prolactin, insulin, and triiodothyronine) differed in lean, insulin-sensitive (3-week-old rats). insulin-resistant (8-week-old rats) and obese, insulin-resistant (44-week-old rats) animals. Simulation of the daily rhythms of endogenous corticosterone and prolactin by daily injections of the hormones at times corresponding to the peak levels found in 3-week-old rats reversed age-related increases in insulin resistance and body fat in older (5-6-month-old) rats. Ten such daily injections of corticosterone and prolactin in 12-14-week-old rats produced long-term reductions in body fat stores (30%). plasma insulin concentration (40%'). and insulin resistance (60%) (determined by a glucose tolerance test) measured 11-14 weeks after the treatment. Alterations in circadian neuroendocrine rhythms may account for age-related changes in carbohydrate and lipid metabolism in the male Sprague-Dawley rat, and resetting of these rhythms by appropriately timed daily injections of corticosterone and prolactin may help maintain metabolism characteristic of younger animals.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

ON THE SITE OF ORIGIN OF TRANSMITTER AMINO ACIDS RELEASED BY DEPOLARIZATION OF NERVE TERMINALS IN VITRO

Abstract— —The site of origin of transmitter amino acids released by depolarizing agents from nerve endings was studied. The model used was the incubated and depolarized synaptosome preparation from which the component soluble, synaptic vesicle, membrane and mitochondrial sub-fractions were obtained. Synaptosomal amino acids were radioactively labelled from D-[U-¹⁴C]glucose in vivo by intraventricular injection and in vitro during subsequent incubation. The specific radioactivities of amino acids released in response to K⁺ (56 mM) or veratrine (75 μM) were found to closely resemble those of the soluble cytoplasmic fraction, in most cases differing significantly from those of the other fractions. The specific radioactivity of the GABA and aspartate released by K⁺ stimulation and the GABA and glutamate released by veratrine were significantly different from that of the vesicles in each case. The specific radioactivities of glutamate released by both agents, and also GABA with K⁺ stimulation, were approximately double that of the amino acid released in control conditions. Depletion of the soluble cytoplasmic pools of glutamate, GABA and aspartate occurred following stimulation, corresponding to the induced-release of these compounds. Turnover of the amino acids in the other subfractions was too low to account for their participation in the release process in addition to the soluble cytoplasmic pool. A cytoplasmic origin of release of neurotransmitter amino acids from nerve endings is proposed.     

Neurotubule assembly at substoichiometric nucleotide levels using a GTP regenerating system

Tubulin derived from cold depolymerized bovine microtubules has been gel filtered to obtain a tubulin preparation with only 3% of the tubulin dimers containing exchangeable [³H]-guanine nucleotide. In the presence of acetyl-P and bacterial acetate kinase, this preparation polymerizes to form microtubules which are morphologically indistinguishable from microtubules formed in the presence of excess GTP. The extent of microtubule formation at substoichiometric nucleotide levels using the GTP regenerating system exceeds the extent of assembly obtained with excess GTP. It is concluded that the exchangeable guanine nucleotide site can be virtually unoccupied in intact neurotubules and this finding indicates that GDP can “catalyze” tubule assembly in the presence of a GTP regenerating system.     

Performance correlates of social behavior and organization: Social rank and complex problem solving in crab-eating macaques (M. fascicularis)

Seventeen male crab-eating macaques, drawn from two captive troops, were tested on a series of complex problem solving tasks in a Wisconsin General Test Apparatus (wgta). The animals were trained on a series of 6-trial object quality learning set problems followed by a series of 10-trial object quality learning set problems. They were then given problems in which the correct stimulus object was reversed part way through the problem. After the animals reached criterion on this task, the reversal learning set was then extinguished. High ranking animals made more intraproblem errors than low ranking animals on the 6-trial problems, but there was no relationship between social status and the rapidity with which the object quality learning set was established. Animals that received overtraining on the 6-trial problems transferred their learning virtually intact to the 10-trial problems; however, high ranking animals without overtraining made more errors than low ranking animals. On reversal learning and reversal extinction, high ranking animals made more errors on critical trials, indicating that they formed and extinguished the reversal set more slowly than low ranking animals. Object quality sets, as measured by trial-2 performance, were not affected by the reversal conditions. Supported by USAMRDC Contract No. DADA 17-73-C-3007.     

An insulin-stimulated kemptide kinase purified from rat liver is deactivated by phosphatase 2A.

Insulin action leads to the rapid stimulation of a cytosolic Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) kinase (KIK) that has been recently purified to near homogeneity (Klarlund, J. K., Bradford, A. P., Milla, M. G., and Czech, M. P. (1990) J. Biol. Chem. 265, 227-234). To examine its activation mechanism, purified KIK was treated with purified protein phosphatases. The catalytic subunit of phosphatase 2A inhibited the activity of control KIK by about 50% and abolished the 5-fold elevation in KIK activity due to insulin action. The catalytic subunit of phosphatase 1 with equivalent activity based on dephosphorylation of 32P-labeled phosphorylase alpha had no effect on either control or insulin-stimulated KIK activity. The deactivation of insulin-stimulated KIK by phosphatase 2A was time- and concentration-dependent and was blocked by phosphatase inhibitors. The purified native complexes of phosphatase 2A, phosphatase 2A1, and phosphatase 2A2 similarly deactivated KIK. Analyis of control or insulin-stimulated KIK with two antiphosphotyrosine antibodies by immunoblotting and immunoprecipitation failed to detect the presence of phosphotyrosine in the kinase. These results indicate that KIK is activated by phosphorylation as part of a kinase cascade emanating from insulin receptor stimulation.     

Human mast cell carboxypeptidase. Selective localization to MCTC cells

Two murine mAb were prepared against human mast cell carboxypeptidase (HMC-CP) purified from human skin, and were termed CP1 and CP2, respectively. Double immunohistochemical labeling of Carnoy's-fixed sections of human skin, lung, and gastrointestinal tissue with CP1 and CP2, respectively, and with a murine monoclonal antitryptase antibody demonstrated that HMC-CP was selectively present in a subset of human mast cells. Double labeling experiments with CP1 and CP2, respectively, and a murine anti-chymase mAb demonstrated the presence of HMC-CP in the tryptase-positive, chymase-positive mast cell type (MCTC) only. Immunohistochemical labeling of peripheral blood leukocytes resulted in staining of monocytes with CP2 but not with CP1. In addition to chymase and a cathepsin-G like proteinase, HMC-CP is another neutral protease that is selectively present in the MCTC tryptase-positive, chymase-positive mast cells type of mast cell, thus extending the biochemical definition of human mast cell heterogeneity.