The metabolic effects of omega-3 plant sterol esters in mixed hyperlipidemic subjects.

PURPOSE: The aim of the current study was to evaluate the therapeutic effects of omega-3 plant sterol esters (n-3-PSE) on lipid profile and other coronary heart disease risk factors in subjects with mixed hyperlipidemia. METHODS: Ninety-one patients with mixed hyperlipidemia were randomized in a double blind fashion to receive either placebo (corn oil) or n-3-PSE. Twenty four patients dropped out or were excluded from the efficacy analysis due to protocol violation. The primary efficacy endpoint was mean change in plasma low-density lipoprotein cholesterol (LDL-C) levels after 12 weeks of treatment. Other efficacy measures included plasma lipids, lipoproteins, and high-sensitivity C-reactive protein (hsCRP) levels. Participants who completed the double-blind study were given the option to continue into an open-label, 12-weeks follow up phase. RESULTS: n-3-PSE treatment did not result in a significant change in LDL-C levels. Triglyceride levels were reduced significantly by 19% (51 mg/dL, p < 0.0001) in the n-3-PSE group in comparison with the placebo group (p = 0.025). Diastolic blood pressure and hsCRP were reduced by 7% (5.9 mmHg) and 7.8% (0.6 mg/L), respectively, and were significantly different from the placebo group (p = 0.036 and p = 0.018, respectively). CONCLUSIONS: In patients with mixed hyperlipidemia, n-3-PSE treatment may offer a safe and effective therapy for triglyceride level reduction while avoiding the typical increase in LDL-C levels associated with n-3 fatty acid treatment. The observed reduction in blood pressure and inflammation markers warrants further evaluation. The positive effect of n-3-PSE treatment was preserved at the end of the follow up phase.

A high oleic sunflower oil fatty acid esters of plant sterols mixed with dietary diacylglycerol reduces plasma insulin and body fat accumulation in Psammomys obesus.

BACKGROUND: Metabolic syndrome is associated with subsequent development of cardiovascular diseases and type 2 diabetes. It is characterized by reduced response to insulin, central obesity, and dyslipidemia. Intake of plant sterols (PS) has been shown to confer a healthier lipid profile and ameliorate cardiovascular disease risk factors in experimental animals and humans. In this study we used an animal model of type 2 diabetes to assess the effects of a preparation of PS esterified to high oleic sunflower oil fatty acids mixed with dietary diacylglycerol (PS-HOSO) on diabetic related metabolic parameters. Psammomys obesus (P. obesus) were fed high energy (HE) diet supplemented by either PS-HOSO or control oil. Following 4.5 weeks of intervention, animals were divided into fasting and non-fasting modes prior to outcome measurements. Glucose and insulin levels as well as blood lipid profile, body weight, and fat accumulation were evaluated in fasting and non-fasting modes. RESULTS: P. obesus fed with a HE diet displayed a characteristic heterogeneity in their blood glucose and insulin levels with a subset group displaying type 2 diabetes symptoms. PS-HOSO treatment significantly reduced total cholesterol (24%, P < 0.001) and non-HDL cholesterol (34%, P < 0.01) compared to the control diet. Among fasting animals, body weight at end point and epididymal fat-to-liver weight ratio were significantly (P < 0.05 each) reduced (7% and 16%, respectively) compared to controls. Interestingly, fasting blood glucose levels were similar between groups, whereas plasma insulin level at end point was 44% lower in the PS-HOSO group compared to control group (P < 0.0001) CONCLUSION: PS-HOSO supplementation to diabetes-prone gerbils counteracts the increase in body weight and epididymal fat accumulation, and also results in a drop in circulating insulin levels. These effects are pointing out that PS-HOSO may serve as a functional ingredient for metabolic syndrome or diabetic sufferers, which not only influences body weight, but also prevents or reverses insulin resistance and hyperlipidemia.

Genome-wide transcriptional plasticity underlies cellular adaptation to novel challenge.

Cells adjust their transcriptional state to accommodate environmental and genetic perturbations. An open question is to what extent transcriptional response to perturbations has been specifically selected along evolution. To test the possibility that transcriptional reprogramming does not need to be 'pre-designed' to lead to an adaptive metabolic state on physiological timescales, we confronted yeast cells with a novel challenge they had not previously encountered. We rewired the genome by recruiting an essential gene, HIS3, from the histidine biosynthesis pathway to a foreign regulatory system, the GAL network responsible for galactose utilization. Switching medium to glucose in a chemostat caused repression of the essential gene and presented the cells with a severe challenge to which they adapted over approximately 10 generations. Using genome-wide expression arrays, we show here that a global transcriptional reprogramming (>1200 genes) underlies the adaptation. A large fraction of the responding genes is nonreproducible in repeated experiments. These results show that a nonspecific transcriptional response reflecting the natural plasticity of the regulatory network supports adaptation of cells to novel challenges.

Synthetic gene recruitment reveals adaptive reprogramming of gene regulation in yeast.

The recruitment of a gene to a foreign regulatory system is a major evolutionary event that can lead to novel phenotypes. However, the evolvability potential of cells depends on their ability to cope with challenges presented by gene recruitment. To study this ability, we combined synthetic gene recruitment with continuous culture and online measurements of the metabolic and regulatory dynamics over long timescales. The gene HIS3 from the histidine synthesis pathway was recruited to the GAL system, responsible for galactose utilization in the yeast S. cerevisiae. Following a switch from galactose to glucose--from induced to repressed conditions of the GAL system--in histidine-lacking chemostats (where the recruited HIS3 is essential), the regulatory system reprogrammed to adaptively tune HIS3 expression, allowing the cells to grow competitively in pure glucose. The adapted state was maintained for hundreds of generations in various environments. The timescales involved and the reproducibility of separate experiments render spontaneous mutations an unlikely underlying mechanism. Essentially all cells could adapt, excluding selection over a genetically variable population. The results reveal heritable adaptation induced by the exposure to glucose. They demonstrate that genetic regulatory networks have the potential to support highly demanding events of gene recruitment.

Regulation of major cellulosomal endoglucanases of Clostridium thermocellum differs from that of a prominent cellulosomal xylanase.

The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CelS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 h(-1), the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 h(-1)) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate-the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.

Regulation of expression of scaffoldin-related genes in Clostridium thermocellum.

Clostridium thermocellum produces an extracellular multienzyme complex, termed the cellulosome, that allows efficient solubilization of crystalline cellulose. The complex is organized around a large noncatalytic protein subunit, termed CipA or scaffoldin, and is found either free in the supernatant or cell bound. The binding of the complex to the cell is mediated by three cell surface anchoring proteins, OlpB, Orf2p, and SdbA, that interact with the CipA scaffoldin. The transcriptional level of the olpB, orf2, sdbA, and cipA genes was determined quantitatively by RNase protection assays in batch and continuous cultures, under carbon and nitrogen limitation. The mRNA level of olpB, orf2, and cipA varied with growth rate, reaching 40 to 60 transcripts per cell under carbon limitation at a low growth rate of 0.04 h(-1) and 2 to 10 transcripts per cell at a growth rate of 0.35 h(-1) in batch culture. The mRNA level of sdbA was about three transcripts per cell and was not influenced by growth rate. Primer extension analysis revealed two major transcriptional start sites, at -81 and -50 bp, upstream of the translational start site of the cipA gene. The potential promoters exhibited homology to the known sigma factors sigma(A) and sigma(L) (sigma(54)) of Bacillus subtilis. Transcription from the sigma(L)-like promoter was found under all growth conditions, whereas transcription from the sigma(A)-like promoter was significant only under carbon limitation. The overall expression level obtained in the primer extension analysis was in good agreement with the results of the RNase-protection assays.

Regulation of the cellulosomal CelS (cel48A) gene of Clostridium thermocellum is growth rate dependent.

Clostridium thermocellum produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose. One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase. The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses indicated that the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures. The transcriptional level of celS mRNA was determined quantitatively by RNase protection assays with batch and continuous cultures under carbon and nitrogen limitation. The amount of celS mRNA transcripts per cell was about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 h(-1) and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 h(-1), respectively. Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 h(-1), respectively. Two major transcriptional start sites were detected at positions -140 and -145 bp, upstream of the translational start site of the celS gene. The potential promoters exhibited homology to known sigma factors (i.e., sigma(A) and sigma(B)) of Bacillus subtilis. The relative activity of the two promoters remained constant under the conditions studied and was in agreement with the results of the RNase protection assay, in which the observed transcriptional activity was inversely proportional to the growth rate.

The eukaryote chaperonin CCT is a cold shock protein in Saccharomyces cerevisiae.

The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide-1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTalpha and CCTbeta, in yeast (Saccharomyces cerevisiae). Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTalpha and CCTbeta genes after cold shock at 4 degrees C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTalpha protein, which is expressed at 10 degrees C, but not at 4 degrees C. Transfer of 4 degrees C cold-shocked cells to 10 degrees C induced a 5-fold increase in the CCTalpha protein level. By means of fluorescent immunostaining and confocal microscopy, we found CCTalpha to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTalpha was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTalpha protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock.