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Background and Purpose: Previous studies have shown that Creosote bush-derived nordihydroguaiaretic acid (NDGA) exerts beneficial actions on the key components of metabolic syndrome including dyslipidaemia, insulin resistance and hypertension in several relevant rodent models. Here, we synthesized and screened a total of 6 anti-hyperlipidaemic analogues of NDGA and tested their efficacy against hepatic lipid metabolism in a high-fructose diet (HFrD) fed dyslipidaemic rat model.Experimental Approach: HFrD fed Sprague-Dawley rats treated with NDGA or one of the six analogues were used. Serum samples were analysed for blood metabolites, whereas liver samples were quantified for changes in various mRNA levels by real-time RT-PCR.Key Results: Oral gavage of HFrD-fed rats for 4 days with NDGA analogues 1 and 2 (100 mg·kg-1 ·day-1 ) suppressed the hepatic triglyceride content, whereas the NDGA analogues 2, 3 and 4, like NDGA, decreased the plasma triglyceride levels by 70-75%. qRT-PCR measurements demonstrated that among NDGA analogues 1, 2, 4 and 5, analogue 4 was the most effective at inhibiting the mRNA levels of some key enzymes and transcription factors involved in lipogenesis. All four analogues almost equally inhibited the key genes involved in triglyceride synthesis and fatty acid elongation. Unlike NDGA, none of the analogues affected the genes of hepatic fatty acid oxidation or transport.Conclusions and Implications: Our data suggest that NDGA analogues 1, 2, 4 and 5, particularly analogue 4, exert their anti-hyperlipidaemic actions by negatively targeting genes of key enzymes and transcription factors involved in lipogenesis, triglyceride synthesis and fatty acid elongation. These analogues have therapeutic potential. [ABSTRACT FROM AUTHOR]

The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt2cAMP-stimulated steroidogenesis ~60–70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging. [ABSTRACT FROM AUTHOR]

The effect of gangliosides and cholera enterotoxin on 125I choriogonadotropin binding, cAMP formation, protein kinase activation and progesterone production have been studied in isolated rat ovarian interstitial cells. A close correlation was observed between the amount of adenosine 3' : 5'-monophosphate (cAMP) produced in the cells and the extent of the activation of protein kinase in response to cholera enterotoxin and choriogonadotropin. Similarly cholera enterotoxin, like choriogonadotropin, stimulated steroidogenesis in a dose-dependent and time-dependent manner. Addition of highly purified ganglioside mixtures abolished the stimulatory effect of cholera enterotoxin on cAMP and progesterone responses, while these agents had no effect on chorio- gonadotropin-stimulated responses. Similar results were also obtained when different lutropin preparations were tested. Pretreatment of ovarian cells with gangliosides did not show any enhancement in the extent of steroid genesis stimulated by toxin or trophic hormones compared to that seen with untreated cells. Both gangliosides and cholera enterotoxin failed to block 125I-labelled choriogonadotropin binding to ovarian cells. Individual gangliosides GD1s, GD1b, GT1 and GM1 [designated according to Svennerholm (1963) J. Neurochem. 10, 613-623) did not modify gonadotropin binding to ovarian cells. Similarly gangliosides isolated from rat ovary, bovine corpus luteum and rat brain also lacked the ability to modify 125I-label led choriogonadotropin binding to cells. Analysis of ovarian cells for gangliosides indicated that major ganglioside was GM3 with trace amounts of other gangliosides including GM1 and more complex gangliosides. The results presented in this communication suggest that gonadotropin binding and subsequent cAMP and progesterone production are not influenced directly by gangliosides. In contrast, choleragen-stimulated cellular events in the ovarian cells were completely inhibited by gangliosides. This suggests that these two agents exert their effects on the responsive system by interacting through separate components on the cell surface. [ABSTRACT FROM AUTHOR]

Phosphodiesterase activities for adenosine and guanosine 3′:5′-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphodiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N′-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 105000 ⊗ g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 μM substrate being 20 μM and 24 μM for cyclic AMP and 7 μM and 10 μM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6–7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+. [ABSTRACT FROM AUTHOR]

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of eyrie [³H]AMP to plasma membranes was increased about 150-200% . Triton X-100 was a better solubilizing agent than Lubrol WX or deoxycholate. In addition to so lubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP stimulation than Ia and IIa peaks. From these studies it is concluded that the plasma-membraneassociated and cytosol protein kinases have sitar catalytic properties bur differ in some of their physical properties. [ABSTRACT FROM AUTHOR]

Investigates the role of cell-surface sialic acid in the interaction of 125 I-labeled choriogonadotropin with gonadotropin receptors from plasma membranes of bovine corpus luteum. Pretreatment of plasma membranes with neuraminidases; Binding activity; Hydrolysis of plasma-membrane sialic acid.