Effects of 2 G on adiposity, leptin, lipoprotein lipase, and uncoupling protein-1 in lean and obese Zucker rats.

Male Zucker rats were exposed to 2 G for 8 wk to test the hypothesis that the leptin regulatory pathway contributes to recovery from effects of 2 G on feeding, growth, and nutrient partitioning. After initial hypophagia, body mass-independent food intake of the lean rats exposed to 2 G surpassed that of the lean rats maintained at 1 G, but food intake of the obese rats exposed to 2 G remained low. After 8 wk at 2 G, body mass and carcass fat were less in both genotypes. Leptin and percent fat were lower in lean rats exposed to 2 G vs. 1 G but did not differ in obese rats exposed to 2 G vs. 1 G. Although exposure to 2 G did not alter uncoupling protein-1 levels, it did elicit white fat pad-specific changes in lipoprotein lipase activity in obese but not lean rats. We conclude that 2 G affects both genotypes but that the lean Zucker rats recover their food intake and growth rate and retain "normal" lipoprotein lipase activity to a greater degree than do the obese rats, emphasizing the importance of a functional leptin regulatory pathway in this acclimation.     

Ventilatory long-term facilitation in unanesthetized rats

Wetested the hypothesis that unanesthetized rats exhibit ventilatorylong-term facilitation (LTF) after intermittent, but not continuous,hypoxia. Minute ventilation (E) and carbon dioxide production (CO₂) were measured inunanesthetized, unrestrained male Sprague-Dawley rats via barometricplethysmography before, during, and after exposure to continuous orintermittent hypoxia. Hypoxia was either isocapnic [inspiredO₂ fraction (FI_O2) = 0.08-0.09 and inspired CO₂ fraction(FI_CO2) = 0.04] or poikilocapnic(FI_O2 = 0.11 andFI_CO2 = 0.00). Sixty minutes afterintermittent hypoxia, E orE/CO₂ wassignificantly greater than baseline in both isocapnic and poikilocapnicconditions. In contrast, 60 min after continuous hypoxia,E andE/CO₂ were notsignificantly different from baseline values. These data demonstrateventilatory LTF after intermittent hypoxia in unanesthetized rats.Ventilatory LTF appeared similar in its magnitude (after accounting forCO₂ feedback), time course, and dependence on intermittenthypoxia to phrenic LTF previously observed in anesthetized,vagotomized, paralyzed rats.

     

Blood pressure response to standing in the diagnosis of autonomic neuropathy: the EURODIAB IDDM Complications Study

Autonomic neuropathy is associated with poor prognosis. Cardiovascular reflexes are essential for the diagnosis of autonomic nerve dysfunction. Blood pressure response to standing is the most simple test for the evaluation of sympathetic integrity, however it is still discussed which diagnostic criteria of abnormal response should be considered as optimal. The EURODIAB IDDM Complications Study involved the examination of randomly selected Type 1 diabetic patients from 31 centres in 16 European counties. Data from 3007 patients were available for the present evaluation. Two tests of autonomic function (response of heart rate /R-R ratio/ and blood pressure from lying to standing) just as the frequency of feeling faint on standing up were assessed. R-R ratio was abnormal in 24% of patients. According to different diagnostic criteria of abnormal BP response to standing (>30 mmHg, >20 mmHg, and >10 mmHg fall in systolic BP), the frequency of abnormal results was 5.9%, 18% and 32%, respectively (p < 0.001). The frequency of feeling faint on standing was 18%, thus, it was identical with the prevalence of abnormal blood pressure response to standing when >20 mmHg fall in systolic blood pressure was considered as abnormal. Feeling faint on standing correlated significantly with both autonomic test results (p < 0.001). A fall >20 mmHg in systolic blood pressure after standing up seems to be the most reliable criterion for the assessment of orthostatic hypotension in the diagnosis of autonomic neuropathy in patients with Type 1 diabetes mellitus.     

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.     

A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA

A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/10(5 )deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 +/- 0.002 for liver to 0.015 +/- 0.003 for brain. We observed a significant increase (10-fold) in oxo8dG in nDNA isolated from liver tissue after 2 Gy of gamma-irradiation when NaI was used to isolate DNA. The turnover of oxo8dG in nDNA was rapid, e.g. disappearance of oxo8dG in the mouse liver in vivo after gamma-irradiation had a half-life of 11 min. The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues. Thus, our results showed that the steady-state levels of oxo8dG in mouse tissues range from 180 to 360 lesions in the nuclear genome and from one to two lesions in 100 mitochondrial genomes.     

Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.     

The influence of lysolipids on the spontaneous curvature and bending elasticity of phospholipid membranes.

The effects of lysolipids on phospholipid layer curvature and bending elasticity were examined using x-ray diffraction and the osmotic stress method. Lysolipids with two different head groups, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and differing hydrocarbon chains were mixed with the hexagonal-forming lipid, dioleoylphosphatidylethanolamine (DOPE). With up to 30 mole% lysolipid in DOPE, the mixture maintains the inverted hexagonal (H(II)) phase in excess water, where increasing levels of lysolipid result in a systematic increase in the H(II) lattice dimension. Analysis of the structural changes imposed by lysolipids show that, opposite to DOPE itself, which has an spontaneous radius of curvature (R(0)) of -30 A, PC lysolipids add high positive curvature, with R(0) = +38 to +60 A, depending on chain length. LysoPEs, in contrast, add very small curvatures. When both polar group and hydrocarbon chains of the added lysolipid mismatch those of DOPE, the structural effects are qualitatively different from otherwise. Such mismatched lysolipids "reshape" the effective combination molecule into a longer and more cylindrical configuration compared to those lysolipids with either matching polar group or hydrocarbon chain.     

A comparison of the metabolic effects of continuous hypothermic perfusion or oxygenated persufflation during hypothermic storage of rat liver

The metabolic consequences of supplying oxygen by two different modes were investigated. The effects of hypothermic liver preservation after cold hypoxic flush (Group I), oxygenated vascular persufflation (Group II), and continuous oxygenated perfusion (Group III) were compared. Adenine nucleotides were measured to assess energetics, and 1H nuclear magnetic resonance spectroscopy was employed to investigate other metabolic pathways. Energetics were maintained by both modes of oxygenation at 24 h. The mitochondrial redox state is indicated by the ratio of acetoacetate (Ace) and beta-hydroxybutyrate (betaHb). The detection of only betaHb or Ace in the hypoxic flush and perfused livers, respectively, suggested that the mitochondria of these livers were hyperreduced and hyperoxidized, respectively. In contrast, both components of the redox couple were detected in the persufflated livers, suggesting that persufflation may be a simple and effective method of maintaining hepatic energetics long-term while maintaining a more normal mitochondrial redox state.