Kelley Strohacker

Pomegranate seed oil consumption during a period of high-fat feeding reduces weight gain and reduces type 2 diabetes risk in CD-1 mice

The health benefits of pomegranate consumption have recently received considerable scientific focus, with most studies examining fruit and/or juice consumption. Pomegranate seed oil (POMo) is a rich source of 9-cis, 11-trans conjugate linolenic acid (CLA), which may offset the side-effects associated with weight gain. Male, wild-type CD-1 mice were divided into one of three groups (twenty per group): high-fat (HF), HF+seed oil (HF + POMo) or lean control (LN). In HF and HF + POMo, mice were provided access ad libitum to a high-fat chow (60 % of energy from fat). HF + POMo was supplemented with 61.79 mg POMo/d. LN consumed a restricted low-fat (10 % of energy from fat) chow to maintain body weight within 5 % of initial weight. Plasma was analysed for biomarkers associated with cholesterol profile (total cholesterol, HDL and TAG), glucose sensitivity (glucose and insulin), adipose tissue accumulation (leptin and adiponectin) and systemic low-grade inflammation (C-reactive protein and haptoglobin). The key findings of this study were that weight gain was associated with an increase in biomarkers of cholesterol profile, glucose sensitivity, adipose tissue accumulation and systemic low-grade inflammation (P < 0.05). POMo only altered body weight accumulation, final body weight, leptin, adiponectin and insulin (P < 0.05). We found that despite a similar level of energy intake, HF mice had a greater concentration of leptin and a lower concentration of adiponectin compared to HF + POMo mice. POMo intake was associated with an improvement in insulin sensitivity, suggesting that risk of developing type 2 diabetes may have been reduced; however, CVD risk did not change.

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters

AIMnOur aim is to investigate the molecular mechanism of regulation of gene expression of drug metabolizing enzymes (DMEs) and transporters in diet-induced obesity.nnnMAIN METHODSnAdult male CD1 mice were fed diets containing 60% kcal fat (HFD) or 10% kcal fat (LFD) for 14 weeks. RNA levels of hepatic DMEs, transporters and their regulatory nuclear receptors (NRs) were analyzed by real-time PCR. Activation of cell-signaling components (JNK and NF-κΒ) and pro-inflammatory cytokines (IL-1β, IL-6 and TNFα) were measured in the liver. Finally, the pharmacodynamics of drugs metabolized by DMEs was measured to determine the clinical relevance of our findings.nnnKEY FINDINGSnRNA levels of the hepatic phase I (Cyp3a11, Cyp2b10, Cyp2a4) and phase II (Ugt1a1, Sult1a1, Sultn) enzymes were reduced ~30-60% in HFD compared to LFD mice. RNA levels of Cyp2e1, Cyp1a2 and the drug transporters, multidrug resistance proteins, (Mrp)2, Mrp3 and multidrug resistant gene (Mdr)1b were unaltered in HFD mice. Gene expression of the NRs, PXR and CAR and nuclear protein levels of RXRα was reduced in HFD mice. Cytokines, JNK and NF-κΒ were induced in HFD mice. Thus reduction in hepatic gene expression in obesity may be modulated by cross-talk between NRs and inflammation-induced cell-signaling. Sleep time of Midazolam (Cyp3a substrate) was prolonged in HFD mice, while Zoxazolamine (Cyp1a2 and Cyp2e1 substrate)-induced sleep time was unaltered.nnnSIGNIFICANCEnThis study demonstrates that gene-specific reductions in DMEs can affect specific drugs metabolized by these enzymes, thus providing a rationale to monitor the effectiveness of drug therapy in obese individuals.

Moderate-intensity, premeal cycling blunts postprandial increases in monocyte cell surface CD18 and CD11a and endothelial microparticles following a high-fat meal in young adults

High-fat meals promote transient increases in proatherogenic factors, implicating the postprandial state in cardiovascular disease (CVD) progression. Although low-grade inflammation is associated with CVD, little research has assessed postprandial inflammation. Because of its anti-inflammatory properties, premeal exercise may counteract postprandial inflammation. The purpose of this study was to determine postprandial alterations in monocytes and circulating markers of endothelial stress and inflammation following a high-fat meal in young adults with or without premeal cycle exercise. Each subject completed two trials and was randomized to rest or cycle at a moderate intensity prior to eating a high-fat meal. Flow cytometry was used to assess monocyte cell surface receptor expression and concentration of endothelial microparticles (EMP). Plasma cytokines were assessed using Luminex MagPix. Statistical analysis was completed using separate linear mixed models analyses with first-order autoregressive (AR(1)) heterogeneous covariance structure. Significance was set at Pxa0≤ 0.05. Percentage increases in classic monocyte CD11a and CD18 were greater overall in the postprandial period in the meal-only condition compared with the mealxa0+ exercise condition (Pxa0< 0.05). EMP concentration was 47% greater 3 h after the meal compared with premeal values in the meal-only condition (Pxa0< 0.05); no significant increase was observed in the mealxa0+ exercise condition. Premeal cycling blunted postprandial increases in EMP and CD11a and CD18. Acute, moderate-intensity exercise may help counteract possibly deleterious postprandial monocyte and endothelial cell activation.

Mouse blood monocytes: standardizing their identification and analysis using CD115.

Monocytes have been used to assess immune dysfunction and disease. While mouse models are a useful longitudinal analog, few researchers have assessed changes in mouse monocytes. The purpose of this study was to provide recommendations for the sample processing and flow cytometric analysis of mouse blood monocytes. Blood was drawn in a non-lethal manner from CD-1 male mice to be used in three experiments. Experiment 1 compared commonly used mouse monocyte markers. Experiment 2 compared the stability of CD115 expression after immediate (0h) and delayed (2 and 4h) processing following blood collection under various experimental conditions (laser strength, anticoagulant, and storage temp.). Experiment 3 compared the consistency of CD115(+) monocyte and subset concentrations using decreasing (40, 20, 10 and 5μL) volumes of blood. In experiment 1, >95% of CD115(+) events co-expressed CD11b; >85% co-expressed CD14. 70% of CD14(+) and 50% of CD11b(+) events co-expressed CD115. In experiment 2, CD115 expression decreased by 33% between 0 and 4h when stored at room temperature. Blood treated with EDTA and refrigerated maintained CD115 stability. In experiment 3, calculated concentrations for total monocyte events varied by <10% when 40, 20 and 10μL of blood were stained. While CD115 staining provides the most distinct monocyte population, it is important to treat blood with EDTA and refrigerate if sample processing will be delayed over 2h. Collectively, the findings of the present study outline important considerations that must be addressed when examining mouse monocytes in small, non-lethal blood samples.

A one-year school-based diet/exercise intervention improves non-traditional disease biomarkers in Mexican-American children.

School-based interventions are an effective way to treat childhood obesity. The purpose of the present study was to biologically validate an established school-based intervention designed to reduce standardised body mass index (zBMI) over a period of 12 months. This intervention focused on a subset of Mexican-American children who were participating in a larger clinical weight loss study. Plasma samples were analysed from self-identified Mexican-American children (12-14 years) who were randomised to either a school-based intervention (IN, n = 152) or self-help control (CN, n = 69). Treatment was 4 days week⁻¹ of exercise (45 min day⁻¹) and 1 day week⁻¹ of nutritional counselling for 6 months. Fasting (>8 h) blood samples were collected at baseline, 6 months (end of active intervention) and 12 months (6 months after the end of the active intervention). Plasma resistin, adiponectin and leptin concentration were measured using a multiplex assay. Separate linear mixed models and a P < 0.05 were used to test for significance. Significant group × time interactions were found for resistin (P < 0.0001), adiponectin (P = 0.001) and leptin (P = 0.013). For resistin, IN was 12% lower at 6 months than CN. Adiponectin concentration in IN was greater at 6 months (26%) and 12 months (8%) than CN. Leptin concentration was 22% lower for IN at 12 months than CN. We have previously reported that our school-based intervention reduced zBMI and now reported alterations in biologically relevant disease biomarkers. Some of the observed changes were only present at the end of the active intervention (resistin), while others persisted until 12 months (leptin and adiponectin). These changes underscore the effectiveness of our school-based intervention at not only improving zBMI but also at reducing disease risk.

Aged mice have increased inflammatory monocyte concentration and altered expression of cell-surface functional receptors

The expression of monocyte cell-surface receptors represents one index of immune dysfunction, which is common with aging. Although mouse models of aging are prevalent, monocyte subset assessment is rare. Our purpose was to compare cell receptor expression on classic (CD115+/Gr-1high) and non-classic (CD115+/Gr-1low) monocytes from 80- or 20-week-old CD-1 mice. Three-colour flow cytometry was used to determine the concentration of monocyte subsets and their respective cell-surface expression of TLR2, TLR4, CD80, CD86, MHC II and CD54. These receptors were selected because they have been previously associated with altered monocyte function. Data were analysed with independent t-tests; significance was set at Pu2009<u20090.05. Old mice had a greater concentration of both classic (258%, Pu2009=u20090.003) and non-classic (70%, Pu2009=u20090.026) monocytes. The classic : non-classic monocyte ratio doubled in old as compared with that in young mice (Pu2009=u20090.006), indicating a pro-inflammatory shift. TLR4 (↓27%, Pu2009=u20090.001) and CD80 (↓37%, Pu2009=u20090.004) were decreased on classic monocytes from old as compared with those from young mice. TLR2 (↑24%, Pu2009=u20090.002) and MHCII (↓21%, Pu2009=u20090.026) were altered on non-classic monocytes from old as compared with those from young mice. The increased classic : non-classic monocyte ratio combined with changes in the cell-surface receptor expression on both monocyte subsets is indicative of immune dysfunction, which may increase age-associated disease risk.

Weight gain in response to high-fat feeding in CD-1 male mice.

The purpose of this study was to compare weight gain and food intake during high-fat feeding in outbred CD-1 male mice while considering several different experimental designs. This study was completed using data from three separate experiments and was designed to address different experimental design issues. Experiment 1 compared mice housed in groups or singly. Experiment 2 compared adolescent and young adult mice. Experiment 3 examined mice that had been previously exercise-trained prior to diet-induced weight gain. Data from each experiment were analysed using repeated measures analysis of variance and linear regression. While housing and age did not significantly affect weight gain, mice that were previously exercise-trained consumed significantly more kilocalories than sedentary mice while maintaining comparable body weights. We generated a linear prediction model using data from Experiments 1 and 2 that will allow investigators to calculate the weeks of high-fat feeding needed to reach a target body weight. Our key findings characterize the issues related to and affecting experimental design when utilizing an outbred mouse diet-induced weight gain model and will serve as a guide for future researchers.

Defining a longitudinal survival model to examine forced treadmill running as a countermeasure for diet-induced weight gain

Diet-induced weight gain increases disease risk via disruption of the innate immune system. Flow cytometry is commonly used to assess the immune system; however, in mice such measurements traditionally require terminal procedures and tissue collection to generate sufficient sample. The present study refined an existing flow cytometry method to reduce the number of mice needed to longitudinally measure monocytes. CD-1 male mice were randomly assigned to one of the three groups: DS (diet-induced weight gain + sedentary), DE (diet-induced weight gain + forced treadmill running [total distance 35,755 ± 1832 m]) or NS (normal weight gain + sedentary). DS and DE consumed a 60% fat diet and NS consumed a 10% fat diet ad libitum. Saphenous vein blood samples were collected weekly for a period of six weeks and three-colour flow cytometry was used to measure changes in monocyte (CD11b+/14+) concentration and cell-surface toll-like receptor 4 (TLR4) expression. DS (18%) and DE (17%) gained more weight than NS (P < 0.001). On a group basis, DS expressed 17% more TLR4 than DE and NS (P = 0.005). The present study demonstrates that a longitudinal survival model can be used to reduce the number of animals needed to complete flow cytometry experiments. Exercise during diet-induced weight prevented some (decreased monocyte TLR4 expression) but not all aspects of innate immune system function.