JNK- and IkappaB-dependent pathways regulate MCP-1 but not adiponectin release from artificially hypertrophied 3T3-L1 adipocytes preloaded with palmitate in vitro

Obese conditions increase the expression of adipocytokine monocyte chemoattractant protein-1 (MCP-1) in adipose tissue as well as MCP-1 plasma levels. To investigate the mechanism behind increased MCP-1, we used a model in which 3T3-L1 adipocytes were artificially hypertrophied by preloading with palmitate in vitro. As observed in obesity, under our model conditions, palmitate-preloaded cells showed significantly increased oxidative stress and increased MCP-1 expression relative to control cells. This increased MCP-1 expression was enhanced by adding exogenous tumor necrosis factor-alpha (TNF-alpha; 17.8-fold vs. control cells, P < 0.01) rather than interleukin-1beta (IL-1beta; 2.6-fold vs. control cells, P < 0.01). However, endogenous TNF-alpha and IL-1beta release was not affected in hypertrophied cells, suggesting that these endogenous cytokines do not mediate hypertrophy-induced increase in MCP-1. MCP-1 secretion from hypertrophied cells was significantly decreased by treatment with antioxidant N-acetyl-cysteine, JNK inhibitors SP600125 and JIP-1 peptide, and IkappaB phosphorylation inhibitors BAY 11-7085 and BMS-345541 (P < 0.01). MCP-1 secretion was not affected by peroxisome proliferator-activated receptor-gamma (PPARgamma) antagonists assayed. Adiponectin, another adipocytokine studied in parallel, also showed increased release in hypertrophy relative to control cells. But in contrast to MCP-1, adiponectin release was significantly suppressed by both exogenous TNF-alpha and IL-1beta as well as by PPARgamma antagonists bisphenol A diglycidyl ether and T0070907 (P < 0.01). JNK inhibitors and IkappaB phosphorylation inhibitors showed no significant effect on adiponectin. We conclude that adipocyte hypertrophy through palmitate loading causes oxidative stress, which in turn increases MCP-1 expression and secretion through JNK and IkappaB signaling. In contrast, the parallel increase in adiponectin expression appears to be related to the PPARgamma ligand properties of palmitate.     

Properties of Crystalline 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum

3β-Hydroxysteroid oxidase (3β-hydroxysteroid: oxygen oxidoreductase, EC 1.1.3.6.) from the culture supernatant of Brevibacterium sterolicum ATCC 21387 has a molecular weight of 32,500 and an isoelectric point of 8.9. The enzyme contained 258 amino acid residues and the composition revealed a distinctive feature of a relatively high amount of proline and the absence of alanine and tryptophan. The crystalline enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 280, 390, and 470 nm with a shoulder at 490 nm. Anaerobic addition of dehydro-epi-androsterone as well as sodium dithionite to the enzyme produced a disappearance of the peaks at 390 and 470 nm. The flavin moiety of the enzyme was isolated and identified as flavin adenine dinucleotide, 1 mole of which was found per mole of protein. The enzyme is sulfhydryl dependent and was inactivated by silver and mercury compounds. Analysis of the enzyme protein by atomic absorption spectrophotometry failed to detect any significant quantity of heavy metals.

Various 3β-hydroxysteroids were oxidized and the relative rates of the oxidation were cholesterol, 100; dehydro-epi-androsterone, 41; pregnenolone, 22; and β-sitosterol, 20. The oxidation product of cholesterol by the enzyme was crystallized and identified as 4-cholesten-3-one by melting point, elementary analysis, optical rotation, UV, IR and NMR spectra. The oxidation of cholesterol proceeded as follows:

The enzyme would be used for some analytical and preparative purposes in the field of steroid chemistry, e.g., microdetermination of cholesterol in serum.     

Comparison of the Effects of Orally Administered Linoleic Acid,and Its Hydroperoxides and Secondary Autoxidation Products on Hepatic Lipid Metabolism in Rats

Linoleic acid, and its hydroperoxides and secondary autoxidation products were orally administered to rats (400 mg/rat). Their effects on hepatic lipid metabolism were examined. Linoleic acid reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase. It decreased the CoASH level and caused the accumulation of long-chain acyl-CoA. Hydroperoxides changed the compositions of unsaturated fatty acids in the hepatic lipids and lowered the content of neutral lipids. Secondary products stimulated carnitine palmitoyltransferase and decreased the content of neutral lipids. They reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase, and the levels of CoASH and acetyl-CoA. Thus, the effect of secondary products was apparently different from those of linoleic acid and its hydroperoxides.     

Basal and Ischemia-Induced Transcardiac Troponin Release into the Coronary Circulation in Patients with Suspected Coronary Artery Disease

Background

Cardiac troponin is a specific biomarker for cardiomyocyte necrosis in acute coronary syndromes. Troponin release from the coronary circulation remains to be determined because of the lower sensitivity of the conventional assay. We sought to determine basal and angina-induced troponin release using a highly sensitive troponin assay.

Methods and Results

The cardiac troponin T levels in serum sampled from the peripheral vein (PV), the aortic root (AO), and the coronary sinus (CS) were measured in 105 consecutive stable patients with coronary risk factor(s) and suspected coronary artery disease (CAD) and in 33 patients without CAD who underwent an acetylcholine provocation test. At baseline, there was a significant increase in the troponin levels from AO [9.0 (6.4, 13.1) pg/mL for median (25^th, 75^th percentiles)] to CS [10.3 (7.3, 15.5) pg/mL, p<0.001] in 96 (91.4%) patients and the difference was 1.1 (0.4, 2.1) pg/mL, which reflected basal transcardiac troponin release (TTR). TTR was positively correlated with PV levels (r = 0.22, p = 0.03). Male sex, left ventricular hypertrophy determined by echocardiography, T-wave inversion, and CAD correlated with elevated TTR defined as above: median, 1.1 pg/mL. A significant increase in TTR was noted in 17 patients with coronary spasms [0.6 (0.2, 1.2) pg/mL, p<0.01] but not in 16 patients without spasms [0.0 (−0.5, 0.9) pg/mL, p = 0.73] after the acetylcholine provocation.

Conclusion

Basal TTR in the coronary circulation was observed in most of the patients with suspected CAD and risk factor(s). This sensitive assay detected myocardial ischemia-induced increases in TTR caused by coronary spasms.     

Why do chlorosomal chlorophylls lack the C132 -methoxycarbonyl moiety? An in vitro model study

Effects of the C13²-methoxycarbonyl moiety on the self-assembly of chlorosomal chlorophylls (Chls) were studied. Model compounds, zinc methyl 3-devinyl-3-(1-hydroxymethyl)-pheophorbides a and a (Zn-3¹-OH-Chls a/a, C13²-epimers) were synthesized from Chl a, and their aggregation behaviors were examined in Triton X-100 (TX-100) micellar suspensions and in 6%THF/water, in comparison with those of a pyrolized derivative, zinc methyl 3-devinyl-3-(1-hydroxymethyl)-13²-demethoxycarbonyl-pheophorbide a (Zn-3¹-OH-pyroChl a). Zn-3¹-OH-Chl a formed self-aggregates in the TX-100 micellar suspension and gave a Q_y absorption peak at 703 nm, while Zn-3¹-OH-pyroChl a aggregates of a Q_y peak at 740 nm. In the Zn-3¹-OH-Chl a aggregate spectrum, the Q_y red-shift was smaller, the band shape was broader, and the contribution of the residual monomer was more intense than that in the Zn-3¹-OH-pyroChl a aggregate spectrum. The bulky C13²-moiety limits the ways of molecular association, and electronic interaction between the component molecules of the Zn-3¹-OH-Chl a aggregate is weakened. Stereoselective control of the aggregation of the C13²-epimer was also examined.     

Protein tyrosine phosphatase LMW-PTP exhibits distinct roles between vascular endothelial and smooth muscle cells

The present study examined the cellular functions of low-molecular-weight protein tyrosine phosphatase (LMW-PTP), which consists of two active isoforms IF-1 and IF-2, in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), focusing on cell growth and migration. We transduced recombinant IF-1 and IF-2, and ribozyme targeting both isoforms using an adenovirus vector in these cells. We detected the expression of IF-1 and IF-2 in both types of cells. IF-1 as well as IF-2 inhibited PDGF-induced DNA synthesis and migration in VSMCs. In contrast, both isoforms enhanced lysophosphatidic acid-stimulated cell migration without change in DNA synthesis in ECs. Whereas there is a report indicating that reactive oxygen species-dependent inactivation of LMW-PTP regulates actin cytoskeleton reorganization during cell spreading and migration, the isoforms conversely suppressed the PDGF-induced H2O2 generation with subsequent decrease in the p38 activity in VSMCs. Catalytically inactive LMW-PTP exerted the opposite and similar effects to the wild type in ECs and in VSMCs, respectively, suggesting that substrates for the phosphatase differ between these cells. Moreover, high concentrations of glucose suppressed the expression of LMW-PTP in both cells. These data suggest that LMW-PTP negatively regulates the pathogenesis of atherosclerosis and that glucose-dependent suppression of LMW-PTP expression may promote the development of atherosclerosis in diabetics.     

Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.