Considerable evidence suggests that the time of day at which calories are consumed markedly impacts body weight gain and adiposity. However, a precise quantification of energy balance parameters ...during controlled animal studies enforcing time-of-day-restricted feeding is currently lacking in the absence of direct human interaction.
The purpose of the present study was therefore to quantify the effects of restricted feeding during the light (sleep)-phase in a fully-automated, computer-controlled comprehensive laboratory animal monitoring system (CLAMS) designed to modulate food access in a time-of-day-dependent manner. Energy balance, gene expression (within metabolically relevant tissues), humoral factors and body weight were assessed.
We report that relative to mice fed only during the dark (active)-phase, light (sleep)-phase fed mice: (1) consume a large meal upon initiation of food availability; (2) consume greater total calories per day; (3) exhibit a higher respiratory exchange ratio (indicative of decreased reliance on lipid/fatty acid oxidation); (4) exhibit tissue-specific alterations in the phases and amplitudes of circadian clock and metabolic genes in metabolically active tissues (greatest phase differences observed in the liver and diminution of amplitudes in epididymal fat, gastrocnemius muscle and heart); (5) exhibit diminished amplitude in humoral factor diurnal variations (for example, corticosterone); and (6) exhibit greater weight gain within 9 days of restricted feeding.
Collectively, these data suggest that weight gain following light (sleep)-phase restricted feeding is associated with significant alterations in energy balance, as well as dyssynchrony between metabolically active organs.
The cardiomyocyte circadian clock directly regulates multiple myocardial functions in a time-of-day-dependent manner, including gene expression, metabolism, contractility, and ischemic tolerance. ...These same biological processes are also directly influenced by modification of proteins by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc). Because the circadian clock and protein O-GlcNAcylation have common regulatory roles in the heart, we hypothesized that a relationship exists between the two. We report that total cardiac protein O-GlcNAc levels exhibit a diurnal variation in mouse hearts, peaking during the active/awake phase. Genetic ablation of the circadian clock specifically in cardiomyocytes in vivo abolishes diurnal variations in cardiac O-GlcNAc levels. These time-of-day-dependent variations appear to be mediated by clock-dependent regulation of O-GlcNAc transferase and O-GlcNAcase protein levels, glucose metabolism/uptake, and glutamine synthesis in an NAD-independent manner. We also identify the clock component Bmal1 as an O-GlcNAc-modified protein. Increasing protein O-GlcNAcylation (through pharmacological inhibition of O-GlcNAcase) results in diminished Per2 protein levels, time-of-day-dependent induction of bmal1 gene expression, and phase advances in the suprachiasmatic nucleus clock. Collectively, these data suggest that the cardiomyocyte circadian clock increases protein O-GlcNAcylation in the heart during the active/awake phase through coordinated regulation of the hexosamine biosynthetic pathway and that protein O-GlcNAcylation in turn influences the timing of the circadian clock.
Circadian dyssynchrony of an organism (at the whole-body level) with its environment, either through light-dark (LD) cycle or genetic manipulation of clock genes, augments various cardiometabolic ...diseases. The cardiomyocyte circadian clock has recently been shown to influence multiple myocardial processes, ranging from transcriptional regulation and energy metabolism to contractile function. The authors, therefore, reasoned that chronic dyssychrony of the cardiomyocyte circadian clock with its environment would precipitate myocardial maladaptation to a circadian challenge (simulated shiftwork; SSW). To test this hypothesis, 2- and 20-month-old wild-type and CCM (Cardiomyocyte Clock Mutant; a model with genetic temporal suspension of the cardiomyocyte circadian clock at the active-to-sleep phase transition) mice were subjected to chronic (16-wks) biweekly 12-h phase shifts in the LD cycle (i.e., SSW). Assessment of adaptation maladaptation at whole-body homeostatic, gravimetric, humoral, histological, transcriptional, and cardiac contractile function levels revealed essentially identical responses between wild-type and CCM littermates. However, CCM hearts exhibited increased biventricular weight, cardiomyocyte size, and molecular markers of hypertrophy (anf, mcip1), independent of aging and or SSW. Similarly, a second genetic model of selective temporal suspension of the cardiomyocyte circadian clock (Cardiomyocyte-specific BMAL1 Knockout CBK mice) exhibits increased biventricular weight and mcip1 expression. Wild-type mice exhibit 5-fold greater cardiac hypertrophic growth (and 6-fold greater anf mRNA induction) when challenged with the hypertrophic agonist isoproterenol at the active-to-sleep phase transition, relative to isoproterenol administration at the sleep-to-active phase transition. This diurnal variation was absent in CCM mice. Collectively, these data suggest that the cardiomyocyte circadian clock likely influences responsiveness of the heart to hypertrophic stimuli. (Author correspondence: meyoung@uab.edu)
The precise biological role of Thy-1, a glycophosphatidyl-inositol (GPI)-linked cell surface glycoprotein in non-caveolar lipid raft microdomains, remains enigmatic. Evidence suggests that Thy-1 ...affects intracellular signaling through src-family protein kinases, and modulates adhesive and migratory events, such as thymocyte adhesion and neurite extension. Primary fibroblasts sorted based on presence or absence of cell surface Thy-1 display strikingly distinct morphologies and differ with respect to production of and response to cytokines and growth factors. It is unclear the extent to which Thy-1 mediates these differences. Findings reported here indicate a novel role for Thy-1 in regulating the activity of Rho GTPase, a critical regulator of cellular adhesion and cytoskeletal organization. Endogenous or heterologous Thy-1 expression promotes focal adhesion and stress fiber formation, characteristic of increased Rho GTPase activity, and inhibits migration. Immunoblotting following transfection of RFL6 fibroblasts with Thy-1 demonstrates that Thy-1 expression inhibits src-family protein tyrosine kinase (SFK) activation, resulting in decreased phosphorylation of p190 Rho GTPase-activating protein (GAP). This results in a net increase in active Rho, and increased stress fibers and focal adhesions. We therefore conclude that Thy-1 surface expression regulates fibroblast focal adhesions, cytoskeletal organization and migration by modulating the activity of p190 RhoGAP and Rho GTPase.
Previous studies suggest that high-density lipoprotein and apoAI inhibit lipopolysaccharide (LPS)-induced inflammatory responses. The goal of the current study was to test the hypothesis that the ...apoAI mimetic peptide L-4F exerts antiinflammatory effects similar to apoAI. Pretreatment of human umbilical vein endothelial cells (HUVECs) with LPS induced the adhesion of THP-1 monocytes. Incubation of cells with LPS and L-4F (1 to 50 microg/mL) reduced THP-1 adhesion in a concentration-dependent manner. This response was associated with a significant reduction in the synthesis of cytokines, chemokines, and adhesion molecules. L-4F reduced vascular cell adhesion molecule-1 expression induced by LPS or lipid A, whereas a control peptide (Sc-4F) showed no effect. In contrast to LPS treatment, L-4F did not inhibit IL-1beta- or tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression. The inhibitory effect of L-4F on LPS induction of inflammatory markers was associated with reduced binding of LPS to its plasma carrier molecule, lipopolysaccharide binding protein, and decreased binding of LPS to HUVEC monolayers. LPS and L-4F in HUVEC culture medium were fractionated by fast protein liquid chromatography and were localized to the same fractions, suggesting a physical interaction between these molecules. Proinflammatory responses to LPS are associated with the binding of lipid A to cell surface receptors. The current studies demonstrate that L-4F reduces the expression of inflammatory markers induced by LPS and lipid A and suggest that apoAI peptide mimetics may be useful in the treatment of inflammation associated with endotoxemia.
The hypothesized relationships between plasminogen activator inhibitor (PAI-1) genotypes, PAI-1 levels, and their potential regulation by hypertriglyceridemic (HTG) very low density lipoprotein ...(VLDL) and lipoprotein(a) Lp(a) was examined in a PAI-1 genotyped human umbilical vein endothelial cell (HUVEC) culture model system. Individual human umbilical veins were used to obtain cultured ECs and were genotyped for PAI-1 by using the HindIII restriction fragment length polymorphism (RFLP) as a marker for genetic variation. Digested genomic DNA, examined by Southern blot analysis and probed with an alpha-32PdCTP-labeled 2.2-kb PAI-1 cDNA, yielded three RFLPs designated 1/1 (22-kb band only), 1/2 (22-plus 18-kb bands), and 2/2 (18-kb band only). Individual PAI-1 genotyped HUVEC cultures were incubated in the absence or presence of HTG-VLDL (0 to 50 micrograms/mL) or Lp(a) (0 to 50 micrograms/mL) at 37 degrees C for various times (4 to 24 hours), followed by analyses of PAI-1 antigen (by ELISA) and mRNA (by ribonuclease protection assay) levels, EC surface-localized plasmin generation assays, and nuclear run-on transcription assays. Secreted PAI-1 antigen levels were increased approximately 2- to 3-fold by HTG-VLDL and approximately 1.6 to 2-fold by Lp(a); mRNA levels were increased approximately 3- to 4.5-fold by HTG-VLDL and approximately 2.5- to 3.2-fold by Lp(a) compared with medium-incubated controls, primarily in the 2/2 PAI-1 genotype HUVEC cultures. Increases in PAI-1 mRNA induced by HTG-VLDL or Lp(a) could be abolished by coincubation with actinomycin D (2 x 10(-6) mol/mL) or puromycin (1 microgram/mL). In addition, nuclear transcription run-on assays typically demonstrated that HTG-VLDL increased PAI-1 gene transcription rates by approximately 5- to 6-fold and approximately 4- to 5-fold, respectively, primarily in the 2/2 PAI-1 genotype HUVEC cultures compared with 1/1 PAI-1 genotype HUVEC cultures or medium-incubated controls. The positive control interleukin-1 increased both 2/2 and 1/1 PAI-1 mRNA levels by approximately 5- to 6-fold. Increased PAI-1 antigen and mRNA expression were associated with a concomitant 50% to 60% decrease in plasmin generation. These combined results demonstrate the genotype-specific regulation of PAI-1 expression by HTG-VLDL and Lp(a) and further indicate that these risk factor-associated components regulate PAI-1 gene expression at the transcriptional level in cultured HUVECs. Results from these studies further suggest that individuals with this responsive 2/2 PAI-1 genotype may reflect the additional inherent potential for later HTG-VLDL- or Lp(a)-induced fibrinolytic dysfunction, resulting in the early initiation of thrombosis, atherogenesis, and coronary artery disease.
Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2ɛ, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of ...plasminogen 2ɛ to CD26 on the cell surface induces a Ca
2+ signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel®. We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2ɛ binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2ɛ. As a result, the plasminogen 2ɛ-induced Ca
2+ signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel® by 1-LN cells is blocked. Angiostatin 2ɛ is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2ɛ may also directly block tumor metastasis.
Wine polyphenol quercetin upregulates tissue-type plasminogen activator (t-PA) transcription in cultured human umbilical cord vein endothelial cells (HUVECs). However, the regulatory elements and ...signaling pathways involved in this regulation are unknown.
We aimed to localize quercetin-responsive t-PA promoter elements, identify the proteins that bind these elements, and decipher signaling pathways involved in the regulation of t-PA.
To localize quercetin-responsive elements, HUVECs were transiently transfected with various t-PA promoter-reporter constructs. Element functionality was evaluated by mutational analysis. Nuclear protein-t-PA element interactions were evaluated by electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) analysis. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the signaling pathways involved in t-PA regulation. MAPK inhibition effects were evaluated by real-time PCR, immunoblotting analysis, and transfections. Coimmunoprecipitation was used to evaluate MAPK and transcription factor interaction.
Deletion of the t-PA promoter region - 288 to - 250 resulted in loss of quercetin responsiveness. This region contains putative Sp1-binding elements, which we termed Sp1a and Sp1b. Sp1b mutation abolished the quercetin-inducible response, whereas Sp1a mutation had no effect. EMSA and ChIP analysis demonstrated quercetin-enhanced Sp1 binding to Sp1b. Inhibition of p38 MAPK abrogated basal and quercetin-induced t-PA expression and promoter activity, as well as quercetin-induced Sp1 binding to Sp1b. Quercetin enhanced p38 MAPK and Sp1 physical association, which was similarly diminished by p38 MAPK inhibition.
We showed, for the first time, the presence of a functional Sp1-binding element in the t-PA promoter controlling quercetin induction via the p38 MAPK pathway. Understanding these mechanisms may provide new insights into polyphenol cardioprotective effects.
Epidemiological evidence indicates that moderate alcohol consumption reduces the incidence of heart disease. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis and ...myocardial functions through the controlled production of nitric oxide (*NO). These studies were conducted to determine if the apparent alcohol-associated cardioprotection is mediated, in part, through modulation of the eNOS protein and activity in the cardiovascular system. Rats were fed alcohol and eNOS protein and *NO production were evaluated at the end of 8 weeks. Myocardial and vascular function was assessed ex vivo in a subset of animals. Moderate alcohol improved postischemic myocardial systolic and diastolic function and attenuated the postischemic reduction in coronary vascular resistance. Moderate alcohol also enhanced maximum vascular relaxation by 26 +/- 0.2% and increased plasma *NO production concomitant with a greater than 2.5-fold increase in eNOS protein. Higher levels of alcohol impaired maximum vascular relaxation by 22 +/- 0.1%. These results suggest that moderate alcohol improves postischemic myocardial functions and increases *NO production by vascular endothelium. An increase in *NO may explain, at least in part, the cardioprotective benefits of moderate alcohol consumption.
Both plasminogen (Pg) activation and matrix metalloproteinases (MMPs) are involved in the proteolytic degradation of extracellular matrix components, a requisite event for malignant cell metastasis. ...The highly invasive 1-LN human prostate tumour cell line synthesizes and secretes large amounts of Pg activators and MMPs. We demonstrate here that the Pg type 2 (Pg 2) receptor in these cells is composed primarily of the membrane glycoprotein dipeptidyl peptidase IV (DPP IV). Pg 2 has six glycoforms that differ in their sialic acid content. Only the highly sialylated Pg 2gamma, Pg 2delta and Pg 2epsilon glycoforms bind to DPP IV via their carbohydrate chains and induce a Ca(2+) signalling cascade; however, Pg 2epsilon alone is also able to significantly stimulate expression of MMP-9. We further demonstrate that the Pg-mediated invasive activity of 1-LN cells is dependent on the availability of Pg 2epsilon. This is the first demonstration of a direct association between the expression of MMP-9 and the Pg activation system.
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