Increased myocardial pyrimidine nucleotide synthesis in isoproterenol-induced cardiac hypertrophy in rats

In a first phase (up to 12^h) after the first injection of isoproterenol (5mg.kg^?1 b.w.) the pyrimidine nucleotide pools were increased and the rates of incorporation of inorganic phosphate into the α-phosphate groups of nucleotides were raised from 16 to 58 nmol.g^?1.h^?1 for uracil nucleotides and from 11 to 32 nmol.g^?1.h^?1 for cytosine nucleotides. At a later stage, while the pool sizes decreased slowly toward control levels, these rates of labelling also decreased though still remaining above control values. A similar pattern of changes was induced by the eighth daily isoproterenol injection, but the alterations were attenuated.     

Increased response to morphine in mice lacking protein kinase C epsilon

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCepsilon have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCepsilon null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCepsilon null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of mu-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCepsilon as a key regulator of opiate sensitivity in mice.     

Phosphorylation of ribosomal and ribosome-associated proteins in isoproterenol-induced cardiac hypertrophy

Cardiac hypertrophy in adult rabbits was induced by subcutaneous injection of isoproterenol. The rate of [3H]leucine incorporation into acid insoluble material was increased and the extent of [32P]phosphate incorporation into several ribosomal proteins was altered. Specifically, a ribosomal protein with a molecular weight of 32,000 from the 40S ribosomal subunit showed a five-fold increase in phosphate incorporation in the hypertrophic heart whereas a protein with a molecular weight of 28,000 from the 60S subunit showed a four-fold decrease. Phosphorylation of ribosome-associated proteins, which could be removed from ribosomes with 0.72 M KCl, was also changed in the hypertrophic hearts. Six major phosphoproteins (with molecular weights 62,000, 49,000, 36,000, 30,000, 20,000 and 12,000) were detected in both the normal and the hypertrophic hearts. Phosphorylation of the 62 K and the 49 K protein was increased by two- and three-fold, respectively, in the hypertrophic hearts, whereas phosphorylation of the 36 K and the 30 K protein decreased by two-fold. The level of phosphorylation of the 20 K and the 12 K protein was not significantly changed in hypertrophic hearts.     

Increased expression of endothelial nitric oxide synthase and caveolin-1 in the aorta of rats with isoproterenol-induced cardiac hypertrophy

Isoproterenol-induced cardiac hypertrophy is associated with increased expression of endothelial nitric oxide synthase in the aorta but without signs of improved endothelial function. The aim was to examine the hypothesis that increased expression of eNOS allosteric inhibitor caveolin-1 could be associated with unimproved endothelium-dependent relaxations. Rats received isoproterenol (5 mg/kg body mass, i.p., n = 13) or its vehicle (n = 14) during 1 week. Systolic blood pressure (SBP) and heart rate (HR) were measured by the tail-cuff method. Expression of eNOS and caveolin-1 was measured using immunoblotting analysis. Relaxations of isolated aorta to acetylcholine and sodium nitroprusside were evaluated ex vivo. After 1 week of isoproterenol administration, basal SBP and HR were decreased (SBP 110 +/- 3 vs. 126 +/- 3 mmHg, p < 0.05; HR 342 +/- 8 vs. 366 +/- 6 beats/min, p < 0.05). Isoproterenol increased the mass of the left ventricle (+33% +/- 4% vs. control; p < 0.05) and right ventricle (+40% +/- 9%; p < 0.05). Isoproterenol administration increased the expression of eNOS (+53% +/- 12%; p < 0.05) and caveolin-1 (+54% +/- 20%, p < 0.05) in the aorta. Relaxation of isolated aorta to acetylcholine and sodium nitroprusside showed a trend towards a worsened endothelial function and a lower sensitivity to exogenous NO. Thus, 1 week of isoproterenol administration led to increased eNOS expression in the aorta without amelioration of endothelial vasorelaxation function. Concomitant increase in caveolin-1 expression may be responsible for this paradox.     

Increased neonatal mortality in mice lacking cellular retinol-binding protein II

Cellular retinol-binding protein II (CRBP II) is a member of the cellular retinol-binding protein family, which is expressed primarily in the small intestine. To investigate the physiological role of CRBP II, the gene encoding CRBP II was inactivated. The saturable component of intestinal retinol uptake is impaired in CRBP II(-/-) mice. The knockout mice, while maintained on a vitamin A-enriched diet, have reduced (40%) hepatic vitamin A stores but grow and reproduce normally. However, reducing maternal dietary vitamin A to marginal levels during the latter half of gestation results in 100% mortality/litter within 24 h after birth in the CRBP II(-/-) line but no mortality in the wild type line. The neonatal mortality in heterozygote offspring of CRBP II(-/-) dams (79 +/- 21% deaths/litter) was increased as compared with the neonatal mortality in heterozygote offspring of wild type dams (29 +/- 25% deaths per litter, p < 0.05). Maternal CRBP II was localized by immunostaining in the placenta at 18 days postcoitum as well as in the small intestine. These studies suggest that both fetal as well as maternal CRBP II are required to ensure adequate delivery of vitamin A to the developing fetus when dietary vitamin A is limiting.     

Dietary cholesterol reverses resistance to diet-induced weight gain in mice lacking Niemann-Pick C1-Like 1

Niemann-Pick C1-Like 1 (NPC1L1) mediates intestinal cholesterol absorption. NPC1L1 knockout (L1-KO) mice were recently shown to be resistant to high-fat diet (HFD)-induced obesity in one study, which was contrary to several other studies. Careful comparison of dietary compositions in these studies implies a potential role of dietary cholesterol in regulating weight gain. To examine this potential, wild-type (WT) and L1-KO mice were fed one of three sets of diets for various durations: (1) a HFD without added cholesterol for 5 weeks; (2) a high-carbohydrate diet with or without added cholesterol for 5 weeks; or (3) a synthetic HFD with or without added cholesterol for 18 weeks. We found that L1-KO mice were protected against diet-induced weight gain only on a diet without added cholesterol but not on a diet containing 0.16% or 0.2% (w/w) cholesterol, an amount similar to a typical Western diet, regardless of the major energy source of the diet. Food intake and intestinal fat absorption were similar between the two genotypes. Intestinal cholesterol absorption was blocked, and fecal cholesterol excretion increased in L1-KO mice. Under all diets, L1-KO mice were protected from hepatosteatosis. In conclusion, increasing dietary cholesterol restores diet-induced weight gain in mice deficient in NPC1L1-dependent cholesterol absorption.     

Prevention of isoproterenol-induced cardiac hypertrophy by beta-blocking agents in the rat.

The effect of the beta-blocking agents propranolol and oxprenolol on isoproterenol-induced cardiac hypertrophy has been investigated in the rat. To evaluate the degree of inhibition biochemical parameters related to cardiomegaly were measured. When given alone in the dose range used for protection, the beta-blocking agents did not cause any significant change in total myocardial RNA, DNA and protein. Depending on the dose applied, propranolol and oxprenolol prevented partially or totally the cardiomegaly induced by isoproterenol.     

Mechanisms of blood pressure variability-induced cardiac hypertrophy and dysfunction in mice with impaired baroreflex

Enhanced blood pressure variability contributes to left ventricular hypertrophy and end-organ damage, even in the absence of hypertension. We hypothesized that the greater number of high-blood pressure episodes associated with enhanced blood pressure variability causes cardiac hypertrophy and dysfunction by activation of mechanosensitive and autocrine pathways. Normotensive mice were subjected to sinoaortic baroreceptor denervation (SAD) or sham surgery. Twelve weeks later, blood pressure variability was doubled in SAD compared with sham-operated mice. Blood pressure did not differ. Cardiac hypertrophy was reflected in greater heart/body weight ratios, larger myocyte cross-sectional areas, and greater left ventricular collagen deposition. Furthermore, left ventricular atrial and brain natriuretic peptide mRNA expression was greater in SAD than in sham-operated mice. SAD had higher left ventricular end-diastolic pressures and lower myocardial contractility indexes, indicating cardiac dysfunction. Cardiac protein content of phosphorylated p125 focal adhesion kinase (p125 FAK) and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) was greater in SAD than in sham-operated mice, indicating activation of mechanosensitive pathways of cardiac hypertrophy. Furthermore, enhanced cardiac renin and transforming growth factor-beta1 (TGFbeta1) protein content indicates activation of autocrine pathways of cardiac hypertrophy. Adrenal tyrosine hydroxylase protein content and the number of renin-positive glomeruli were not different, suggesting that sympathetic activation and the systemic renin-angiotensin system did not contribute to cardiac hypertrophy. In conclusion, more frequent blood pressure rises in subjects with high blood pressure variability activate mechanosensitive and autocrine pathways leading to cardiac hypertrophy and dysfunction even in the absence of hypertension.     

Enhanced platelet activation mediates the accelerated angiogenic switch in mice lacking histidine-rich glycoprotein

Background

The heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice.

Methodology/Principal Findings

To address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG^−/− mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG^+/+ littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice.

Conclusions

Our data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.     

Overconsumption and obesity: peptides and susceptibility to weight gain

Physiological control of feeding is mediated by tonic and episodic signalling systems. These are sometimes thought of as long-term and short-term control. Tonic signals arise from tissue stores whereas episodic signals oscillate periodically with the consumption of food. These physiological controls are paralleled in the motivation to eat by long-acting enduring traits (such as disinhibition) and by short-acting states (such as hunger). Peptides are usually envisaged to exert an action on appetite control through the modulation of states such as hunger and satiety (fullness). Here we provide evidence that peptides involved in tonic regulation--such as leptin--may express a control over appetite motivation through an effect on traits that confer a constant readiness to eat, whereas episodic peptides such as GLP-1 influence appetite motivation through a state such as hunger. The distinction between tonic and episodic regulation, and between traits and states has implications for understanding overconsumption and the susceptibility to weight gain.     

Increased fat mass and insulin resistance in mice lacking pancreatic lipase-related protein 1

Pancreatic triglyceride lipase (PTL) and its cofactor, colipase, are required for efficient dietary triglyceride digestion. In addition to PTL, pancreatic acinar cells synthesize two pancreatic lipase-related proteins (PLRP1 and PLRP2), which have a high degree of sequence and structural homology with PTL. The lipase activity of PLRP2 has been confirmed, whereas no known triglyceride lipase activity has been detected with PLRP1 up to now. To explore the biological functions of PLRP1 in vivo, we generated Plrp1 knockout (KO) mice in our laboratory. Here we show that the Plrp1 KO mice displayed mature-onset obesity with increased fat mass, impaired glucose clearance and the resultant insulin resistance. When fed on high-fat (HF) diet, the Plrp1 KO mice exhibited an increased weight gain, fat mass and severe insulin resistance compared with wild-type mice. Pancreatic juice extracted from Plrp1 KO mice had greater ability to hydrolyze triglyceride than that from the wild-type littermates. We propose that PLRP1 may function as a metabolic inhibitor in vivo of PLT-colipase-mediated dietary triglyceride digestion and provides potential anti-obesity targets for developing new drugs.     

Increase in type I adenosine 3',5'-monophosphate-dependent protein kinase during isoproterenol-induced cardiac hypertrophy.

The amount of type I and type II cyclic AMP-dependent protein kinase present in the rat heart was determined at various times during isoproterenol-induced cardiac hypertrophy. Wistar rats were injected twice daily with isoproterenol (5 mg/kg, s.c.) for 2, 5 or 10 days. Cardiac weight increased gradually over the 10-day period of drug administration, and by day 10, heart weight was 156% of control. Following the cessation of isoproterenol administration, the cardiac weight regressed toward the control value by day 15. An increase in the specific activity of type I protein kinase to 197% of control occurred by day 10. The specific activity of type II protein kinase did not change significantly during either the hypertrophy or regression stage. The increase in the specific activity of type I protein kinase during a chemically-induced trophic response of the heart may indicate that type I cyclic AMP-dependent protein kinase plays a regulatory function in this process.     

Increased susceptibility of ST2-deficient mice to polymicrobial sepsis is associated with an impaired bactericidal function

ST2, a member of the Toll/IL-1R superfamily, negatively regulates both TLR2 and TLR4 signaling. In this study, we report that ST2-deficient mice were more susceptible to polymicrobial sepsis than their wild-type littermates, with increased production of proinflammatory cytokines. Bacterial clearance from the circulation and visceral organs following polymicrobial infection was markedly impaired in ST2-deficient mice. This was associated with substantially reduced uptake, phagocytosis, and intracellular killing of both Gram-positive and Gram-negative bacteria by ST2-deficient phagocytes. Consistent with a reduced antimicrobial response, phagocytes lacking ST2 displayed a defect in bactericidal activity in response to bacterial challenges with severely impaired phagosome maturation and NOX2 function. Thus, ST2-deficient mice exhibit an increased susceptibility to polymicrobial infection with impaired bacterial clearance, which is associated with defects in phagosome maturation and NOX2-derived production of reactive oxygen species characterized in ST2-deficient phagocytes.     

Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics.