A black soybean seed coat extract prevents obesity and glucose intolerance by up-regulating uncoupling proteins and down-regulating inflammatory cytokines in high-fat diet-fed mice.

Black soybean seed coat extract (BE) is a polyphenol-rich food material consisting of 9.2% cyanidin 3-glucoside, 6.2% catechins, 39.8% procyanidins, and others. This study demonstrated that BE ameliorated obesity and glucose intolerance by up-regulating uncoupling proteins (UCPs) and down-regulating inflammatory cytokines in C57BL/6 mice fed a control or high-fat diet containing BE for 14 weeks. BE suppressed fat accumulation in mesenteric adipose tissue, reduced the plasma glucose level, and enhanced insulin sensitivity in the high-fat diet-fed mice. The gene and protein expression levels of UCP-1 in brown adipose tissue and UCP-2 in white adipose tissue were up-regulated by BE. Moreover, the gene expression levels of major inflammatory cytokines, tumor necrosis factor-α and monocyte chemoattractant protein-1 were remarkably decreased by BE in white adipose tissue. BE is a beneficial food material for the prevention of obesity and diabetes by enhancing energy expenditure and suppressing inflammation.

Impact of a metabolic uncoupler, 2,4-dichlorophenol on minimization of activated sludge production in membrane bioreactor.

This study investigated the effects of 2,4-dichlorophenol on reduction of activated sludge in membrane bioreactors. Significant inhibition on sludge growth and slight reduction in COD removal were observed at higher 2,4-dichlorophenol dosages. The deviation between relative specific COD removal rate (q/q0) and relative specific growth rate (µ/µ0) suggested that a minimum 2,4-dichlorophenol concentration was required for uncoupling of anabolism and catabolism. With the increase of the dosage of 2,4-dichlorophenol, stepwise improvement of biomass bioactivity and the reduction in activated sludge production were achieved simultaneously. Compared with the control bioreactor, the peak distribution of floc size in the 2,4-dichlorophenol added bioreactor shifted to a range of smaller floc size. Besides, addition of 2,4-dichlorophenol caused little variation of microorganism community structure and SVI value of the sludge. After 24-hour operation, the residue 2,4-dichlorophenol concentration in the bioreactors was reduced to a negligible level.

Determination of partition coefficient and analysis of nitrophenols by three-phase liquid-phase microextraction coupled with capillary electrophoresis.

A three-phase hollow fiber liquid-phase microextraction method coupled with CE was developed and used for the determination of partition coefficients and analysis of selected nitrophenols in water samples. The selected nitrophenols were extracted from 14 mL of aqueous solution (donor solution) with the pH adjusted to pH 3 into an organic phase (1-octanol) immobilized in the pores of the hollow fiber and finally backextracted into 40.0 microL of the acceptor phase (NaOH) at pH 12.0 located inside the lumen of the hollow fiber. The extractions were carried out under the following optimum conditions: donor solution, 0.05 M H(3)PO(4), pH 3.0; organic solvent, 1-octanol; acceptor solution, 40 microL of 0.1 M NaOH, pH 12.0; agitation rate, 1050 rpm; extraction time, 15 min. Under optimized conditions, the calibration curves for the analytes were linear in the range of 0.05-0.30 mg/L with r(2)>0.9900 and LODs were in the range of 0.01-0.04 mg/L with RSDs of 1.25-2.32%. Excellent enrichment factors of up to 398-folds were obtained. It was found that the partition coefficient (K(a/d)) values were high for 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol and 2,6-dinitrophenol and that the individual partition coefficients (K(org/d) and K(a/org)) promoted efficient simultaneous extraction from the donor through the organic phase and further into the acceptor phase. The developed method was successfully applied for the analysis of water samples.

Effect of calcium-enriched high-fat diet on calcium, magnesium and zinc retention in mice.

The aim of this work was to assess the effects of a high-fat diet enriched in Ca, which accompanies lower body fat deposition, on mineral depots, as well as to assess the potential role of adaptive thermogenesis in mice. Male mice were fed ad libitum a high-fat (43 %) diet with a Ca content of 4 g/kg from calcium carbonate (control group) or 12 g/kg (42 % from milk powder and the rest from calcium carbonate) (Ca group) for 56 d. Body weight, food intake and urine were periodically collected. Tissue samples were collected when the mice were killed and the composition was determined. Expression of uncoupling proteins was determined by Western blotting. Mineral content was measured by flame atomic absorption spectrometry. Lower body weight gain and fat accretion was found in the Ca group. This could not be attributable to lower gross energy intake or to activation of adaptive thermogenesis. Although significant urine mineral loss was found in the Ca group, preservation of mineral depots in bone was observed. Our data support the fact that adding more Ca to the diet, using a combination of calcium carbonate plus milk powder containing among other things higher Zn and Mg, contributes to counteracting obesity and improving lipid metabolism.

Effects of exercise and obesity on UCP3 content in rat hindlimb muscles.

UNLABELLED: Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein, which is hypothesized to shuttle nonmetabolized fatty acids, particularly when excessive fatty acids are present. PURPOSE: Obese Zucker rats (OZR) have systematically elevated levels of fatty acids, with decreased fatty acid metabolism. We hypothesized that basal UCP3 protein expression levels would be elevated in the skeletal muscles of the OZR compared with the lean Zucker rats (LZR). In addition, because aerobic exercise training has been shown to elevate the ability of skeletal muscle to metabolize lipids, we also hypothesized that aerobic exercise training would decrease skeletal muscle UCP3 protein expression and that this would be more pronounced in the skeletal muscles of the OZR. METHODS: OZR and LZR were aerobically trained on a motorized treadmill for 55 min x d(-1), 5 d x wk(-1), for 9 wk. UCP3 and oxidative enzymes were measured in plantaris, gastrocnemius, and soleus muscles. RESULTS: Basal UCP3 protein expression was elevated approximately eightfold in the plantaris muscles and threefold in the gastrocnemius muscles of the OZR compared with the LZR (P < 0.05). However, there was no difference in UCP3 protein expression in the soleus muscles of the OZR compared with the LZR (P = 0.34). Furthermore, aerobic exercise training did not significantly alter UCP3 protein expression in the soleus, plantaris, or gastrocnemius muscles of the LZR; however, UCP3 protein expression levels decreased in trained OZR soleus and gastrocnemius muscles compared with controls. CONCLUSIONS: The decrease in UCP3 with aerobic exercise training was most notable in the soleus of the OZR. These data demonstrate that the exercise-induced adaptations of UCP3 protein levels are muscle specific in obese animals compared with lean animals.

Pediatric fatality following ingestion of dinitrophenol: postmortem identification of a "dietary supplement".

Dinitrophenol, a chemical currently used as an insecticide, is known to uncouple mitochondrial oxidative phosphorylation. A component of explosives, it has also been used in the past as a food coloring and clothing dye. In the 1930s, physicians prescribed it for weight loss, but this practice was discontinued when reports of cataracts, deaths, and other adverse outcomes came to light. We describe in our report the overdose and fatality of a teenager who purchased the product as a weight loss dietary supplement by mail order. We also describe a laboratory method that allowed postmortem determination of the dinitrophenol concentration in the victim's serum. Her death, despite prompt medical treatment, underscores the danger of dinitrophenol. The easy accessibility and apparent resurgent interest in dinitrophenol as a weight loss agent is extremely timely and troubling.

Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle.

Uncoupling proteins (UCPs) present in the inner mitochondrial membrane are involved in uncoupling respiration from ATP synthesis. Five UCP isoforms have been identified but information about their presence and level of expression in the central nervous system remains incomplete. To determine the nature and proportion of UCP isoform mRNAs present in brain cortex, we developed and optimized a specific quantitative reverse-transcription polymerase chain reaction procedure. Optimal range of RNA concentrations to be used in the reverse-transcriptase reaction was determined. Primer design and concentration were optimized for each target gene while polymerase chain reaction efficiency was assessed for a range of reverse-transcriptase dilutions. Genomic contribution to the quantitative signal was evaluated for each isoform and minimized. Three reference genes were tested for normalization, and beta-actin was found to be the most stable among tissues. Results indicate that brain cortex contains significant amounts of all UCP mRNAs, with UCP5 and UCP4 being the most abundant, as opposed to brown adipose tissue and skeletal muscle, which predominantly express UCP1 and UCP3, respectively. These data provide a first quantitative assessment of UCP mRNA expression in mouse brain, showing the presence of all five isoforms with distinct proportions, thus suggesting specific roles in the central nervous system.

Improved glucose homeostasis in mice overexpressing human UCP3: a role for AMP-kinase?

OBJECTIVE: An unexplained phenotype of mice overexpressing human UCP3 is their improved glucose homeostasis. Since overexpression of UCP3 might affect the energy charge of the cell, we investigated whether these mice have an increased AMP-activated protein kinase (AMPK) activity. METHODS: Mitochondrial localisation of UCP3 was determined by immunoelectronmicroscopy and AMPK activity was measured in medial gastrocnemius of control mice and mice overexpressing human UCP3. RESULTS: Mice overexpressing human UCP3 had 5.8 fold higher levels of UCP3 protein, for which mitochondrial localisation was confirmed by immunoelectronmicroscopy. The ATP/AMP ratio was significantly lower in mice over-expressing UCP3 compared to the wild-type (10.9+/-1.6 vs 20.4+/-1.9 AU, P=0.03). Over-expression of UCP3 resulted in increased AMPK alpha1 activity (1.23+/-0.05 vs 1.00+/-0.06 normalized values, P=0.004) and a tendency towards increased AMPK alpha2 activity (1.18+/-0.08 vs 1.00+/-0.10 normalized values, P=0.08). CONCLUSION: Increased AMPK activity provides a plausible explanation for the improved glucose tolerance characteristic for these mice.

Detection of DNA fragmentation and apoptotic proteins, and quantification of uncoupling protein expression by real-time RT-PCR in adipose tissue.

Better understanding of the mechanisms involved in adipose tissue growth and metabolism is critical for the development of more effective treatments for obesity. However, because of its high lipid and low protein content, adipose tissue can present unique problems in some experimental procedures. We describe three protocols that provide new or improved methods for analysis of DNA, RNA, and protein from different adipose tissues. The first protocol provides a simple and rapid method for separation of fragmented DNA and visualization of apoptotic DNA laddering without the need for radioisotopes. This technique allows for an estimate of the amount of DNA fragmentation, and hence, apoptosis. The second protocol details subcellular fractionation of adipose tissue for the extraction of protein in the mitochondrial and cytosol fractions and the measurement of apoptotic protein (Bcl-2 and Bax) levels in each fraction. The last protocol involves extraction of total RNA from adipose tissue and the measurement of uncoupling protein mRNA using real-time RT-PCR, a method that has not previously been used to measure expression of uncoupling proteins in adipose tissue.

The effect of mild cold exposure on UCP3 mRNA expression and UCP3 protein content in humans.

OBJECTIVE: In rodents, adaptive thermogenesis in response to cold exposure and high-fat feeding is accomplished by the activation of the brown adipose tissue specific mitochondrial uncoupling protein, UCP1. The recently discovered human uncoupling protein 3 is a possible candidate for adaptive thermogenesis in humans. In the present study we examined the effect of mild cold exposure on the mRNA and protein expression of UCP3. SUBJECTS: Ten healthy male volunteers (age 24.4 +/- 1.6 y; height 1.83 +/- 0.02 m; weight 77.3 +/- 3.0 kg; percentage body fat 19 +/- 2). DESIGN: Subjects stayed twice in the respiration chamber for 60 h (20.00-8.00 h); once at 22 degrees C (72 degrees F), and once at 16 degrees C (61 degrees F). After leaving the respiration chamber, muscle biopsies were taken and RT-competitive-PCR and Western blotting was used to measure UCP3 mRNA and protein expression respectively. RESULTS: Twenty-four-hour energy expenditure was significantly increased at 16 degrees C compared to 22 degrees C (P<0.05). At 16 degrees C, UCP3T (4.6 +/- 1.0 vs 7.7 +/- 1.5 amol/microg RNA, P=0.07), UCP3L (2.0 +/- 0.5 vs 3.5 +/- 0.9 amol/microg RNA, P=0.1) and UCP3S (2.6 +/- 0.6 vs 4.2 +/- 0.7 amol/microg RNA, P=0.07) mRNA expression tended to be lower compared with at 22 degrees C, whereas UCP3 protein content was, on average, not different. However, the individual differences in UCP3 protein content (16-22 degrees C) correlated positively with the differences in 24 h energy expenditure (r=0.86, P<0.05). CONCLUSION: The present study suggests that UCP3 protein content is related to energy metabolism in humans and might help in the metabolic adaptation to cold exposure. However, the down-regulation of UCP3 mRNA with mild cold exposure suggests that prolonged cold exposure will lead to lower UCP3 protein content. What the function of such down-regulation of UCP3 could be is presently unknown.

Toxicity of 2,6-Di-tert-butyl-4-Nitrophenol (DBNP).

U.S. Navy submarines reported a yellowing of metal surfaces on their internal surfaces. The yellowing was initially identified on the painted steel bulkheads but further examination indicated that it was not limited to steel surfaces and included bedding, thread tape, Formica, plastisol covered hand-wheels, and aluminum lockers. Crew members also reported to the medical department that their skin turned yellow when they came in contact with these contaminated surfaces and requested information on the effects of exposure. Studies conducted by General Dynamics' Electric Boat Division (EBD) determined that the agent was 2,6-Di-tertbutyl-4-Nitrophenol (DBNP). 2,6-Di-butylphenol (DBP) is an antioxidant additive used in lubricating oils and hydraulic fluids. In the enclosed atmosphere of a submarine, the oil mist could be spread throughout the boat by venting the lube oil to the atmosphere. Submarines use electrostatic precipitators (ESP) to clean the air of particulate materials. During passage through the ESP, oil mist containing DBP is nitrated to DBNP, which is then moved throughout the boat in the ventilation system. Analysis of the EBD data indicated 24-hour exposure concentrations to be in the range of <3.0 to 122 ppb in the laboratory and submarine settings. Submarine crews may be exposed to these concentrations for as many as 24 hours/ day for 90 days during underway periods. Toxicity studies regarding the oral and dermal uptake of DBNP were conducted. From the literature the lethal dose to 50 percent of the population (LD50) of DBNP (rat) was reported by Vesselinovitch et al. in 1961 to be 500 mg/kg. Our studies indicated that the LD50 is in the range of 80 mg/kg in the rat. Our work also includes dermal absorption studies, which indicated that DBNP is not well absorbed through intact skin. Within this study, no no-observable adverse effect level (NOAEL) or lowest observable adverse effect level (LOAEL) was identified. Calculation of a reference dose was completed using standard methods based on the LD50 as a numerator with several uncertainty and modifying factors. EBD's determination of airborne concentrations aboard submarines fall in the range of these anticipated allowable concentrations and could indicate significant chronic exposures. No adverse effects from DBNP exposures have been reported to date.

The effect of weight reduction on skeletal muscle UCP2 and UCP3 mRNA expression and UCP3 protein content in Type II diabetic subjects.

AIMS/HYPOTHESIS: The aim of this study was to examine the effect of weight loss on UCP2/UCP3 mRNA expression and UCP3 protein content in subjects with Type II (non-insulin-dependent) diabetes mellitus. METHODS: We studied seven Type II diabetic subjects who followed a 10-week very low calorie diet. Expression of skeletal muscle UCP2 and UCP3 mRNA was measured using RT-competitive PCR and UCP3 protein content by western blotting, before and after the diet. Total and plasma fatty acid oxidation was measured using infusion of 13C labelled palmitate. RESULTS: Body weight decreased from 105.5 +/- 8.2 kg to 91.6 +/- 7.2 kg (p < 0.001), after 10 weeks of diet intervention. Expression of UCP2 and UCP3 mRNA were significantly reduced after 10 weeks of diet (p < 0.05) but UCP3 protein contents were not significantly altered. Notably, the change in UCP3L mRNA expression and UCP3 protein content after the very low calorie diet were negatively associated with changes in body weight (r = -0.97, p = 0.006 and r = -0.83, p = 0.043, respectively) and BMI (r = -0.99, p = 0.0007 and r = -0.9, p = 0.016, respectively). Furthermore, changes in UCP3L mRNA expression and UCP3 protein content induced by the diet were positively correlated with changes in cytosolic fatty acid-binding protein content (r = 0.93, p = 0.023 and r = 0.84, p = 0.039, respectively). No correlation between diet-induced changes in UCP3 protein and resting energy expenditure or plasma non-esterified fatty acid concentrations were found. CONCLUSION/INTERPRETATION: The negative correlation between the change in UCP3 protein content after weight loss and the change in BMI, suggests that the decrease in UCP3 during weight loss could prevent further weight loss. The finding that the change in UCP3 protein content correlates with the change in skeletal muscle fatty acid-binding protein content, suggests a role for UCPs in the handling of lipids as a fuel.

Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression.

Leptin is believed to act through hypothalamic centers to decrease appetite and increase energy utilization, in part through enhanced thermogenesis. In this study, we examined the effects of fasting for 2 days and exogenous s.c. leptin, 200 microg every 8 h for 2 days, on the regulation of uncoupling protein (UCP) subtypes in brown adipose tissue (BAT) and gastrocnemius muscle. Northern blot analysis (UCP-1) and ribonuclease protection (UCP-2 and 3) were used for quantitative messenger RNA (mRNA) analysis, and specific antibodies were used to measure UCP-1 and UCP-3 total protein expression. Leptin, compared with vehicle, did not alter BAT UCP-1 or UCP-3 mRNA or protein expression when administered to normal ad libitum fed rats. Fasting significantly decreased BAT UCP-1 and UCP-3 mRNA expression, to 31% and 30% of ad libitum fed controls, respectively, effects which were prevented by administration of leptin to fasted rats. Fasting also significantly decreased BAT UCP-1 protein expression, to 67% of control; however, that effect was not prevented by leptin treatment. Fasting also decreased BAT UCP-3 protein, to 85% of control, an effect that was not statistically significant. Fasting, with or without leptin administration, did not affect BAT UCP-2 mRNA; however, leptin administration to ad libitum fed rats significantly increased BAT UCP-2 mRNA, to 138% of control. Fasting significantly enhanced gastrocnemius muscle UCP-3 mRNA (411% of control) and protein expression (168% of control), whereas leptin administration to fasted rats did not alter either of these effects. In summary, UCP subtype mRNA and protein are regulated in tissue- and subtype-specific fashion by leptin and food restriction. Under certain conditions, the effects of these perturbations on UCP mRNA and protein are discordant.

Analysis of uncoupling protein and its mRNA in adipose tissue deposits of adult humans.

Brown adipose tissue (BAT) is a specialized adipose tissue whose specific marker is the uncoupling protein (UCP). UCP and its mRNA were previously detected in the perirenal fat of several adult subjects undergoing surgery for pheochromocytoma. We have investigated the possible association of the presence of UCP and its mRNA with pathological conditions other than pheochromocytoma. We obtained adipose tissue from both the periadrenal and the perirenal regions of 36 subjects: group A: human infants (n = 6); group B: adult subjects (n = 11) undergoing surgery for pheochromocytoma; group C: adult subjects (n = 9) undergoing surgery for other endocrine pathologies; group D: adult patients (n = 10) operated for non-endocrine pathologies. In all subjects of group A UCP was detectable by Western analysis. Interestingly, in two newborns, we also found a positive signal for UCP in the peristernal and the retroperitoneal adipose tissues as well as in the perirenal fat. We also identified UCP in eight cases in group B, in five cases in group C and six cases in group D. The human H-UCP-0.5 genomic probe detected a typical BAT mRNA in the periadrenal adipose tissue of all subjects of groups B, C and D showing a positive Western blot. Our results confirm the presence of well-developed BAT in human infants, as well as in adults with pheochromocytoma. They also suggest that human BAT UCP and UCP mRNA are present in adult subjects in pathological conditions other than pheochromocytoma. It might be argued that certain hormones distinct from catecholamine could activate BAT development in human adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of brown adipose tissue uncoupling protein by enzyme linked immunosorbent assay.

Antibody to uncoupling protein (UCP) purified from rat brown adipose tissue (BAT) was raised in rabbits and an enzyme linked immunosorbent assay was developed. The antiserum did not cross-react with other mitochondrial proteins from BAT and from other tissues but cross-reacted with UCP from hamster, guinea pig and mouse. The assay is capable of detecting 5 ng of UCP. Using this assay and a crude mitochondrial preparation, UCP content of BAT was shown to increase during cold adaptation.

Postnatal appearance of uncoupling protein and formation of thermogenic mitochondria in hamster brown adipose tissue.

Brown adipose tissue of developing hamster was characterized by western blotting, enzyme activity measurements and immunoelectron microscopy. During the first postnatal week the tissue contained significant amounts of differentiating mitochondria and comparable quantities of active cytochrome oxidase and ATP synthase. The uncoupling protein appeared on the 7/8th day and its specific content increased 80-times between day 8 and day 17. In parallel, the specific content and activity of cytochrome oxidase increased 3-times but ATP synthase decreased 2-times. The total content of uncoupling protein and of cytochrome oxidase in interscapular brown adipose tissue increased 360- and 11-times, respectively. Analysis of isolated mitochondria showed that the observed differences result mainly from changes of the enzymic equipment of the mitochondrial membrane. During the same interval, propylthiouracil-insensitive "type II' thyroxine 5'-deiodinase activity in brown adipose tissue increased 10-times. It was concluded that the thermogenic function of the hamster brown adipose tissue develops after the first postnatal week due to highly differentiated synthesis of mitochondrial proteins leading to replacement of preexisting, uncoupling protein-lacking nonthermogenic mitochondria by thermogenic ones, similarly as shown in brown adipose tissue of the embryonic mouse and rat (Houstek, J., et al. (1988) Biochim. Biophys. Acta 935, 19-25).

The novel thermogenic beta-adrenergic agonist Ro 16-8714 increases the interscapular brown-fat beta-receptor-adenylate cyclase and the uncoupling-protein mRNA level in obese (fa/fa) Zucker rats.

In obese (fa/fa) rats both the total (-)-isoprenaline- and NaF-stimulated adenylate cyclase activities of interscapular brown-adipose-tissue (IBAT) plasma membranes were decreased as compared with lean rats by 40 and 38% respectively. An acute treatment with Ro 16-8714 increased (-)-isoprenaline- and NaF-stimulated adenylate cyclase activities by 2.5- and 2.0-fold respectively, and beta-adrenoceptor number 2.8-fold in obese rat IBAT, but it had no effect on these parameters in lean rats. It increased mRNA for mitochondrial uncoupling protein in both lean and obese rats to the same extent.

Sulfhydryl groups of the uncoupling protein of brown adipose tissue mitochondria. Distinction between sulfhydryl groups of the H+ channel and the nucleotide binding site.

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.