Role of Mitochondrial Uncoupling Protein 2 (UCP2) in Modulation of Adiposity

Obesity is a disorder of energy balance in which energy intake exceeds energy expenditure. Methods to control obesity through limiting energy intake have had limited success, and it is widely recognized that energy expenditure must also be increased if long-term weight loss is to be achieved. Uncoupling proteins (UCPs) are a family of integral membrane proteins of mitochondrial inner membrane, where they uncouple the process of mitochondrial respiration from oxidative phosphorylation, diminishing the resulting production of ATP and decreasing the metabolic efficiency of the organism. Thus, UCPs provide new molecular targets for increasing energy expenditure. Unlike the other UCP family members, UCP2 is ubiquitously expressed, with the highest level in white adipose tissue. Stimulation of mitochondrial uncoupling in adipocytes in vitro demonstrates a direct inhibitory effect on lipogenesis and suppression on lipolysis via a calcium dependent mechanism, indicating a potential role of UCP2 in regulation of adiposity. Previous studies demonstrate that intracellular Ca2+ ([Ca2+]i ) plays a key role in metabolic disorders associated with obesity. [Ca2+]i can clearly be modulated by the calcitrophic hormone 1α, 25-dihydroxyvitamin D3 (1α, 25-(OH)2-D3), which appears to have both genomic and non-genomic action in adipocytes. 1α, 25-(OH)2-D3 modulates adipocyte Ca2+ signaling directly, resulting in an increased lipogenesis and decreased lipolysis. In addition, 1α, 25-(OH)2-D3 plays a genomic role in regulating adipocyte UCP2 expression levels, indicating that the regulation of UCP2 and the resulting increased core temperature may contribute to increased rates of energy dissipation. Accordingly, the suppression of 1α, 25-(OH)2-D3 by increasing dietary calcium attenuates adiposity by decreasing triglyceride accumulation in the adipocytes: increasing dietary calcium results in a net reduction in fat mass in the absence of caloric restriction, a marked augmentation of body weight and fat loss during energy restriction, and an inhibition of weight and fat regain after food restriction in mice. Although these anti-obesity effects of dietary calcium are due, in part, to enhanced metabolic rate and thermogenic processes, it is also possible that a loss of adipocytes would result in a deficit cells for lipid esterification as the body recovers. Physiological doses of 1α, 25-(OH)2-D3 inhibit apoptosis in differentiated 3T3-L1 adipocytes, and the suppression of 1α, 25-(OH)2-D3 in vivo by increasing dietary calcium stimulates adipocyte apoptosis in refeeding following energy restriction in aP2 transgenic mice, indicating that the stimulation of adipocyte apoptosis contributes to adiposity reduction after high calcium diet administration. UCP2 plays a direct role in modulating adipocyte apoptosis by inducing mitochondrial potential collapse and inhibiting ATP production. Overexpression of UCP2 in adipocyte stimulates apoptosis while inhibition of mitochondrial uncoupling either by chemical inhibitor GDP or by siRNA duplexes suppresses adipocyte apoptosis. Accordingly, suppression of UCP2 by physiologically low doses of 1α, 25-(OH)2-D3, which can be induced by low dietary calcium, decreases apoptosis. Although 1α, 25-(OH)2-D3 causes dose-dependent stimulation on [Ca2+]i levels, low doses of 1α, 25-(OH)2-D3 decrease mitochondrial calcium accumulation while a high dose of 1α, 25-(OH)2-D3 induces markedly greater increase in [Ca2+]i and stimulates calcium storage in mitochondria. Bay K 8644, which is a Ca2+ ionophore and can mimic the stimulatory effect of 1α, 25-(OH)2-D3 on [Ca2+]i without exerting effects on UCP2, causes a dose-dependent increases in apoptosis and mitochondrial calcium accumulation, indicating that stimulation of pharmaceutical high dose 1α, 25-(OH)2-D3 on apoptosis is a calcium-dependent effect. In summary, present data support the concept that dietary calcium exerts antiobesity effects in aP2 transgenic mice under conditions of varying nutrient status. In addition, this study extended our observation that dietary calcium not only regulates adipocyte size by decreasing lipid accumulation, but also modulates adipocyte number by stimulating apoptotic death. These anti-obesity effects of dietary calcium are attributable to the up-regulation of UCP2, which stimulates energy expenditure, fat utilization and adipocyte apoptosis in white adipose tissue. Accordingly, these data indicate an important role of increasing dietary calcium in prevention and management of obesity

Leucine modulation of mitochondrial mass and oxygen consumption in skeletal muscle cells and adipocytes

<p>Abstract</p> <p>Background</p> <p>The effects of dairy on energy metabolism appear to be mediated, in part, by leucine and calcium which regulate both adipocyte and skeletal muscle energy metabolism. We recently demonstrated that leucine and calcitriol regulate fatty acid oxidation in skeletal muscle cells <it>in vitro</it>, with leucine promoting and calcitriol suppressing fatty acid oxidation. Moreover, leucine coordinately regulated adipocyte lipid metabolism to promote flux of lipid to skeletal muscle and regulate metabolic flexibility. We have now investigated the role of mitochondrial biogenesis in mediating these effects.</p> <p>Methods</p> <p>We tested the effect of leucine, calcitriol and calcium in regulation of mitochondrial mass using a fluorescence method and tested mitochondrial biogenesis regulatory genes as well mitochondrial component genes using real-time PCR. We also evaluated the effect of leucine on oxygen consumption with a modified perfusion system.</p> <p>Results</p> <p>Leucine (0.5 mM) increased mitochondrial mass by 30% and 53% in C2C12 myocytes and 3T3-L1 adipocytes, respectively, while calcitriol (10 nM) decreased mitochondrial abundance by 37% and 27% (p < 0.02). Leucine also stimulated mitochondrial biogenesis genes SIRT-1, PGC-1α and NRF-1 as well as mitochondrial component genes UCP3, COX, and NADH expression by 3–5 fold in C2C12 cells (p < 0.003). Adipocyte-conditioned medium reduced mitochondrial abundance (p < 0.001) and decreased UCP3 but increased PGC-1α expression in myocytes, suggesting a feedback stimulation of mitochondrial biogenesis. Similar data were observed in C2C12 myocytes co-cultured with adipocytes, with co-culture markedly suppressing mitochondrial abundance (p < 0.02). Leucine stimulated oxygen consumption in both C2C12 cells and adipocytes compared with either control or valine-treated cells. Transfection of C2C12 myocytes with SIRT-1 siRNA resulted in parallel suppression of SIRT-1 expression and leucine-induced stimulation of PGC-1α and NRF-1, indicating that SIRT-1 mediates leucine induced mitochondrial biogenesis in muscle cells.</p> <p>Conclusion</p> <p>These data suggest that leucine and calcitriol modulation of muscle and adipocyte energy metabolism is mediated, in part, by mitochondrial biogenesis.</p

Cortisol, progesterone, 17α-hydroxyprogesterone, and TSH responses in dogs injected with low-dose lipopolysaccharide

Background. Stress and diseases such as endotoxemia induce cortisol synthesis through a complex biosynthetic pathway involving intermediates (progesterone, and 17αhydroxyprogesterone (17α-OHP)) and suppression of the hypothalamus-pituitarythyroid axis. Objective. To measure plasma concentrations of cortisol, progesterone, 17α-OHP, and thyroid stimulating hormone (TSH) in dogs experimentally injected with intravenous low-dose lipopolysaccharide (LPS). Our hypothesis was that LPS treatment would elicit a significant increase in cortisol and its precursors, and a significant decrease in TSH concentration. Methods. Hormone measurements were performed on blood samples left over from a previous investigation (2011) on the effect of low-dose LPS on hematological measurands. Five sexually intact female dogs, none in estrous at the time of the study, were administered saline treatment two weeks prior to LPS treatment. LPS was administered intravenously at a dose of 0.1 µg/kg. Blood was collected before (baseline, time -24 hours) and 3-, 6- and 24-hours post-injection. Mixed model analysis for repeated measures was used, with both treatment and time as the repeated factors. Ranked transformation were applied when diagnostic analysis exhibited violation of normality and equal variance assumptions. Post hoc multiple comparisons were performed with Tukey’s adjustment. Statistical significance was defined as p \u3c 0.05. Results. Significant differences relative to baseline values were detected following both treatments. Compared to baseline, dogs had significantly higher cortisol and 17α-OHP at 3-hours, and significantly lower TSH at 3- and 6-hours following LPS treatment. Dogs had significantly lower TSH at 6- and 24- following saline treatment. Though not statistically significant, the trend in progesterone concentrations was similar to cortisol and 17α-OHP, with an increase at 3-hours post-injection followed by a decrease close to baseline following both LPS and saline. Cortisol and 17α-OHP concentrations were higher after LPS treatment than after saline treatment at 3- and 6-hours post-injection, but differences were not statistically significant, and no significant differences between treatments were detected for any other hormone or timepoint

Characterization of the Fecal Microbiome in Dogs Receiving Medical Management for Congenital Portosystemic Shunts

BackgroundThe GI microbiome has not been characterized in dogs being medically managed for congenital portosystemic shunts (CPSS).ObjectivesTo characterize the fecal microbiome in a population of dogs being medically managed for CPSS.Animals27 client-owned dogs.MethodsProspective cohort study enrollment of fecal samples was performed with follow-up data collected retrospectively. The overall fecal dysbiosis index (DI) and individual bacterial abundances were determined using real-time qPCR. Medical management, clinical findings, clinicopathologic, and outcome variables were collected, and logistic regression analyses were performed to evaluate associations between these variables and overall DI and bacterial abundances. Numerical variables were evaluated with general linear models for normality and equal variance using Shapiro-Wilk test and Levene's test, respectively.ResultsAll dogs were administered a hepatic diet and lactulose, while antibiotics were used in 22 (81.5%) and acid suppressants in 7 (25.9%). Seventeen dogs (63.0%) had a DI &gt;2. The median DI in this population was 3.02 (range 4.23–8.42), and the median DI in dogs receiving and not receiving antibiotics was 4.3 (range −4.23–8.42) and 1.52 (range −1.62–5.43), respectively. No significant association between any of the analyzed variables and the DI was identified. There was a significant association between the use of metronidazole and a larger abundance of E. coli (p = 0.024).Conclusions and Clinical ImportanceDysbiosis appears to be common in dogs that are being medically managed for CPSS, though the clinical significance remains unclear

Effects of dairy intake on weight maintenance

Background: To compare the effects of low versus recommended levels of dairy intake on weight maintenance and body composition subsequent to weight loss. Design and Methods: Two site (University of Kansas-KU; University of Tennessee-UT), 9 month, randomized trial. Weight loss was baseline to 3 months, weight maintenance was 4 to 9 months. Participants were maintained randomly assigned to low dairy ( 3 servings/d) diets for the maintenance phase. Three hundred thirty eight men and women, age: 40.3 ± 7.0 years and BMI: 34.5 ± 3.1, were randomized; Change in weight and body composition (total fat, trunk fat) from 4 to 9 months were the primary outcomes. Blood chemistry, blood pressure, resting metabolism, and respiratory quotient were secondary outcomes. Energy intake, calcium intake, dairy intake, and physical activity were measured as process evaluation. Results: During weight maintenance, there were no overall significant differences for weight or body composition between the low and recommended dairy groups. A significant site interaction occurred with the low dairy group at KU maintaining weight and body composition and the low dairy group at UT increasing weight and body fat. The recommended dairy group exhibited reductions in plasma 1,25-(OH)2-D while no change was observed in the low dairy group. No other differences were found for blood chemistry, blood pressure or physical activity between low and recommended dairy groups. The recommended dairy group showed significantly greater energy intake and lower respiratory quotient compared to the low dairy group. Conclusion: Weight maintenance was similar for low and recommended dairy groups. The recommended dairy group exhibited evidence of greater fat oxidation and was able to consume greater energy without greater weight gain compared to the low dairy group. Recommended levels of dairy products may be used during weight maintenance without contributing to weight gain compared to diets low in dairy products. Trial Registration: ClinicalTrials.gov NCT0068642

Effects of dairy intake on weight maintenance

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Coal char effects on soil chemical properties and maize yields in semi-arid region

Soil amendments with high carbon (C) content can be effective in semi-arid regions where soils are characterized by low C. A field study was conducted in 2016–2018 to evaluate the effect of char on soil chemical properties and irrigated maize (Zea mays L.) yields in sandy loam fertilized with urea or composted manure. Carbon-rich char used was a product of coal combustion residue from a local factory in western Nebraska. The experiment was arranged in a split-plot randomized complete block design in four replications with char (0, 6.7, 13.4, 20.1, and 26.8Mg C ha−1) as main and N treatment (0, 90, 180, and 270 kg urea-N ha−1 and 33.6 and 67.2 Mg ha−1 of composted manure) as subplot factors. A handheld spectral sensor was used to determine normalized difference red edge (NDRE) at growth stages (V6, V8, V10, and R1) in 2017 and 2018. After 2 yr, char increased Fe, reduced pH at lower rates, and increased K and Mg at higher rates in top 20 cm soil but did not affect crop yields. Char applied at ≥13.4 Mg C ha−1 increased soil organic C by ≥8% and composted manure increased soil P and K compared to the control. There was a strong correlation of NDRE with N rates and grain yields at V8 and V10. This study found no adverse effect of char on soil properties. However, more site-specific research is needed before char can be used as a regular soil amendment in semi-arid regions

The effectiveness of cooling conditions on temperature of canine EDTA whole blood samples

Background Preanalytic factors such as time and temperature can have significant effects on laboratory test results. For example, ammonium concentration will increase 31% in blood samples stored at room temperature for 30 min before centrifugation. To reduce preanalytic error, blood samples may be placed in precooled tubes and chilled on ice or in ice water baths; however, the effectiveness of these modalities in cooling blood samples has not been formally evaluated. The purpose of this study was to evaluate the effectiveness of various cooling modalities on reducing temperature of EDTA whole blood samples. Methods Pooled samples of canine EDTA whole blood were divided into two aliquots. Saline was added to one aliquot to produce a packed cell volume (PCV) of 40% and to the second aliquot to produce a PCV of 20% (simulated anemia). Thirty samples from each aliquot were warmed to 37.7 °C and cooled in 2 ml allotments under one of three conditions: in ice, in ice after transfer to a precooled tube, or in an ice water bath. Temperature of each sample was recorded at one minute intervals for 15 min. Results Within treatment conditions, sample PCV had no significant effect on cooling. Cooling in ice water was significantly faster than cooling in ice only or transferring the sample to a precooled tube and cooling it on ice. Mean temperature of samples cooled in ice water was significantly lower at 15 min than mean temperatures of those cooled in ice, whether or not the tube was precooled. By 4 min, samples cooled in an ice water bath had reached mean temperatures less than 4 °C (refrigeration temperature), while samples cooled in other conditions remained above 4.0 °C for at least 11 min. For samples with a PCV of 40%, precooling the tube had no significant effect on rate of cooling on ice. For samples with a PCV of 20%, transfer to a precooled tube resulted in a significantly faster rate of cooling than direct placement of the warmed tube onto ice. Discussion Canine EDTA whole blood samples cool most rapidly and to a greater degree when placed in an ice-water bath rather than in ice. Samples stored on ice water can rapidly drop below normal refrigeration temperatures; this should be taken into consideration when using this cooling modality

The effect of postoperative oral antibiotic therapy on the incidence of postoperative endophthalmitis after phacoemulsification surgery in dogs: 368 eyes (1997–2010)

Purpose To assess the effectiveness of postoperative administration of oral antibiotics at reducing the incidence of endophthalmitis following phacoemulsification cataract extraction in dogs. Methods Medical records of the University of Tennessee College of Veterinary Medicine were reviewed for cases having undergone phacoemulsification and divided according to whether or not they had received oral antibiotics postoperatively. Records were then evaluated for a diagnosis of endophthalmitis and incidence rates between the group receiving postoperative oral antibiotics and the group not receiving postoperative oral antibiotics were compared. Results A total of 215 patients (368 eyes) were identified by the search. One-hundred twelve patients (197 eyes) were treated with oral antibiotics postoperatively. One-hundred and three patients (171 eyes) were not treated with oral antibiotics postoperatively. Three cases of endophthalmitis were identified, with one in the antibiotic-treated group and two in the non-antibiotic treated group (P > 0.05, Fisher’s exact test). Conclusions The overall incidence of endophthalmitis at the University of Tennessee from 1997–2010 was 0.82%. The rate of post-phacoemulsification endophthalmitis was unaffected by the postoperative administration of oral antibiotics