Epigenetic modification of miR-663 controls mitochondria-to-nucleus retrograde signaling and tumor progression.

The normal cellular function requires communication between mitochondria and the nucleus, termed mitochondria-to-nucleus retrograde signaling. Disruption of this mechanism has been implicated in the development of cancers. Many proteins are known modulators of retrograde signaling, but whether microRNAs (miRNAs) are also involved is unknown. We conducted an miRNA microarray analysis using RNA from a parental cell line, a Rho0 line lacking mitochondrial DNA (mtDNA) and a Rho0 line with restored mtDNA. We found that miR-663 was down-regulated in the mtDNA-depleted Rho0 line. mtDNA restoration reversed this miRNA to parental level, suggesting that miR-663 may be epigenetically regulated by retrograde signaling. By using methylation-specific PCR and bisulfite sequencing we demonstrate that miR-663 promoter is epigenetically regulated not only by genetic but also by pharmacological disruption of oxidative phosphorylation (OXPHOS). Restoration of OXPHOS Complex I inhibitor-induced miR-663 expression by N-acetylcysteine suggested that reactive oxygen species (ROS) play a key role in epigenetic regulation of miR-663. We determined that miR-663 regulates the expression of nuclear-encoded respiratory chain subunits involved in Complexes I, II, III, and IV. miR-663 also controlled the expression of the Complexes I (NDUFAF1), II (SDHAF2), III (UQCC2), and IV (SCO1) assembly factors and was required for stability of respiratory supercomplexes. Furthermore, using luciferase assays, we found that miR-663 directly regulates UQCC2. The anti-miR-663 reduced OXPHOS complex activity and increased in vitro cellular proliferation and promoted tumor development in vivo in mice. We also found that increased miR-663 expression in breast tumors consistently correlates with increased patient survival. We provide the first evidence for miRNA controlling retrograde signaling, demonstrating its epigenetic regulation and its role in breast tumorigenesis.

Dietary Plant Sterol Esters Must Be Hydrolyzed to Reduce Intestinal Cholesterol Absorption in Hamsters.

BACKGROUND: Elevated concentrations of LDL cholesterol are associated with the development of atherosclerosis and therefore are considered an important target for intervention to prevent cardiovascular diseases. The inhibition of cholesterol absorption in the small intestine is an attractive approach to lowering plasma cholesterol, one that is addressed by drug therapy as well as dietary supplementation with plant sterols and plant sterol esters (PSEs). OBJECTIVE: This study was conducted to test the hypothesis that the cholesterol-lowering effects of PSE require hydrolysis to free sterols (FSs). METHODS: Male Syrian hamsters were fed atherogenic diets (AIN-93M purified diet containing 0.12% cholesterol and 8% coconut oil) to which one of the following was added: no PSEs or ethers (control), 5% sterol stearate esters, 5% sterol palmitate esters (PEs), 5% sterol oleate esters (OEs), 5% sterol stearate ethers (STs; to mimic nonhydrolyzable PSE), or 3% FSs plus 2% sunflower oil. The treatments effectively created a spectrum of PSE hydrolysis across which cholesterol metabolism could be compared. Metabolic measurements included cholesterol absorption, plasma and liver lipid concentration, and fecal neutral sterol and bile acid excretion. RESULTS: The STs and the PEs and SEs were poorly hydrolyzed (1.69-4.12%). In contrast, OEs were 88.3% hydrolyzed. The percent hydrolysis was negatively correlated with cholesterol absorption (r = -0.85; P < 0.0001) and positively correlated with fecal cholesterol excretion (r = 0.92; P < 0.0001), suggesting that PSE hydrolysis plays a central role in the cholesterol-lowering properties of PSE. CONCLUSIONS: Our data on hamsters suggest that PSE hydrolysis and the presence of FSs is necessary to induce an optimum cholesterol-lowering effect and that poorly hydrolyzed PSEs may lower cholesterol through an alternative mechanism than that of competition with cholesterol for micelle incorporation.

Diet-induced alterations of host cholesterol metabolism are likely to affect the gut microbiota composition in hamsters.

The gastrointestinal microbiota affects the metabolism of the mammalian host and has consequences for health. However, the complexity of gut microbial communities and host metabolic pathways make functional connections difficult to unravel, especially in terms of causation. In this study, we have characterized the fecal microbiota of hamsters whose cholesterol metabolism was extensively modulated by the dietary addition of plant sterol esters (PSE). PSE intake induced dramatic shifts in the fecal microbiota, reducing several bacterial taxa within the families Coriobacteriaceae and Erysipelotrichaceae. The abundance of these taxa displayed remarkably high correlations with host cholesterol metabolites. Most importantly, the associations between several bacterial taxa with fecal and biliary cholesterol excretion showed an almost perfect fit to a sigmoidal nonlinear model of bacterial inhibition, suggesting that host cholesterol excretion can shape microbiota structure through the antibacterial action of cholesterol. In vitro experiments suggested a modest antibacterial effect of cholesterol, and especially of cholesteryl-linoleate, but not plant sterols when included in model bile micelles. The findings obtained in this study are relevant to our understanding of gut microbiota-host lipid metabolism interactions, as they provide the first evidence for a role of cholesterol excreted with the bile as a relevant host factor that modulates the gut microbiota. The findings further suggest that the connections between Coriobacteriaceae and Erysipelotrichaceae and host lipid metabolism, which have been observed in several studies, could be caused by a metabolic phenotype of the host (cholesterol excretion) affecting the gut microbiota.

Food availability of glucose and fat, but not fructose, increased in the U.S. between 1970 and 2009: analysis of the USDA food availability data system.

BACKGROUND: Obesity rates in the United States have risen consistently over the last four decades, increasing from about 13% of the population in 1970 to more than 34% in 2009. Dietary fructose has been blamed as a possible contributor to the obesity increase, although the consumption pattern of fructose and other key nutrients during this 40 year period remains a topic of debate. Therefore, we analyzed the USDA Loss-Adjusted Food Availability Database in combination with the USDA Nutrient Database for Standard Reference (Release 24) to determine whether fructose consumption in the US has increased sufficiently to be a casual factor in the rise in obesity prevalence. METHODS: Per capita loss-adjusted food availability data for 132 individual food items were compiled and analyzed. Nutrient profiles for each of these foods were used to determine the availability of energy as well as macronutrients and monosaccharides during the years 1970-2009. The percent change in energy from food groups and individual nutrients was determined by using the year 1970 as the baseline and area-under-the-curve analysis of food trends. RESULTS: Our findings indicate that during this 40 year period the percent change in total energy availability increased 10.7%, but that the net change in total fructose availability was 0%. Energy available from total glucose (from all digestible food sources) increased 13.0%. Furthermore, glucose availability was more than 3-times greater than fructose. Energy available from protein, carbohydrate and fat increased 4.7%, 9.8% and 14.6%, respectively. CONCLUSIONS: These data suggest that total fructose availability in the US did not increase between 1970 and 2009 and, thus, was unlikely to have been a unique causal factor in the increased obesity prevalence. We conclude that increased total energy intake, due to increased availability of foods providing glucose (primarily as starch in grains) and fat, to be a significant contributor to increased obesity in the US.

Unrefined and refined black raspberry seed oils significantly lower triglycerides and moderately affect cholesterol metabolism in male Syrian hamsters.

Unrefined and refined black raspberry seed oils (RSOs) were examined for their lipid-modulating effects in male Syrian hamsters fed high-cholesterol (0.12% g/g), high-fat (9% g/g) diets. Hamsters fed the refined and the unrefined RSO diets had equivalently lower plasma total cholesterol and high-density lipoprotein (HDL) cholesterol in comparison with the atherogenic coconut oil diet. The unrefined RSO treatment group did not differ in liver total and esterified cholesterol from the coconut oil-fed control animals, but the refined RSO resulted in significantly elevated liver total and esterified cholesterol concentrations. The unrefined RSO diets significantly lowered plasma triglycerides (46%; P=.0126) in comparison with the coconut oil diet, whereas the refined RSO only tended to lower plasma triglyceride (29%; P=.1630). Liver triglyceride concentrations were lower in the unrefined (46%; P=.0002) and refined (36%; P=.0005) RSO-fed animals than the coconut oil group, with the unrefined RSO diet eliciting a lower concentration than the soybean oil diet. Both RSOs demonstrated a null or moderate effect on cholesterol metabolism despite enrichment in linoleic acid, significantly lowering HDL cholesterol but not non-HDL cholesterol. Dramatically, both RSOs significantly reduced hypertriglyceridemia, most likely due to enrichment in α-linolenic acid. As a terrestrial source of α-linolenic acid, black RSOs, both refined and unrefined, provide a promising alternative to fish oil supplementation in management of hypertriglyceridemia, as demonstrated in hamsters fed high levels of dietary triglyceride and cholesterol.