The experimental procedures and protocols of this study conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.
The ability of CO inhalation to either prevent or reverse obesity was studied using separate groups exposed to 2 different concentrations of CO. CO inhalation was performed in a specially designed chamber which allowed mice to be exposed to different levels of CO without leaving their home cages. Two different levels of CO inhalation exposures were used in the present study. Mice were exposed to CO at 28 parts per million (ppm) for 2 hours daily (CO-28) or 200 ppm CO for 1 hour daily (CO-200). All mice were housed under standard conditions with full access to a 60% high fat diet (diet # D12492, Research Diets, Inc., New Brunswick, NJ) and water. Control mice were fed the high fat diet but did not did not receive any CO exposure. For the prevention study, a separate cohort of mice was maintained on a 17% normal fat diet (Teklad 22/5 rodent diet, #8640, Harland Laboratories, Inc., Indianapolis, IN).
Mice were exposed to CO in a 25 × 27 × 38 cm clear plastic chamber. A mixture of CO and air was flushed through the chamber at a rate of 0.05–30 l/min. Pure CO gas was mixed with appropriate amounts of air to dilute the CO to the desired concentration in parts per million (ppm), either ~28 or 200 ppm CO. A sampling line was fed from the chamber and passed through a Grey Wolf Indoor Air IQ-410 CO gas analyzer (Shelton, CT) which was connected to a lap-top computer and gave real time CO gas measurements using the WolfSense software package (Version 2011.28, Shelton, CT). The CO concentration was verified using a handheld CO meter placed inside the inhalation chamber (C.E., China). This setup allowed for mice to be exposed to the different levels of CO with free access to food and water in their home cages throughout the daily treatment periods. Mice were exposed to CO in the mornings for 2 hours in mice receiving 28 ppm CO and for 1 hour in mice receiving 200 ppm.
Carboxyhemoglobin (COHb) measurement
Using a separate group of mice from those used in the chronic CO inhalation studies; COHb was measured before, immediately after and at 30, 60 and 90 minutes following each CO inhalation exposure protocol. Blood was collected via the orbital sinus under light isoflurane anesthesia. Immediately after collection, the blood sample was analyzed for COHb levels using Radiometer ABL80 Flex CO-OX analyzer (Westlake, OH) which requires 105 μl of whole blood. COHb levels are expressed as a percentage of total hemoglobin.
Body composition (EchoMRI)
Body composition of all mice was assessed at 6 week intervals throughout the study using magnetic resonance imaging (EchoMRI-900TM, Echo Medical System, Houston, TX). EchoMRI measurements were performed in conscious mice placed in a thin-walled plastic cylinder with a cylindrical plastic insert added to limit movement of the mice. Mice were briefly submitted to a low intensity electromagnetic field allowing both fat mass and lean mass to be measured.
Following an overnight fast (~16 hours) a blood sample was obtained under isoflorane anesthesia via orbital sinus. Blood glucose was measured using an Accu-Chek Advantage glucometer (Roche, Mannheim, Germany).
Oxygen consumption, carbon dioxide production and motor activity. Twenty-eight weeks after the initiation the experimental protocol 4 mice from each group were placed individually in an acrylic cage (16 cm × 24 cm × 17 cm) equipped with a metabolic monitoring system (AccuScan system, Harvard Apparatus, Holliston, Massachusetts) for measurements of oxygen consumption (VO2), carbon dioxide production (VCO2) and respiratory quotient (RQ) as previously described [15, 16]. VO2, VCO2 and RQ were determined daily (for 2 min every 10-min interval) and expressed as the 24 hour average. RQ was calculated by the formula: VCO2/VO2. Motor activity was determined using infrared light beams mounted in the cages in x, y, and z axes. Heat production was derived from the following formula (4.33 + (0.67*RQ)*VO2*weight (g)*60). After the mice were acclimatized to the new environment for 1 day, VO2, VCO2, RQ and animal activity were recorded for 2 consecutive days.
Glucose tolerance test
For glucose tolerance tests (GTT), mice were subjected to an overnight fast (∼16 h) and intraperitoneal (IP) injection of D-glucose (1 g/kg of body weight). Blood glucose was monitored at 0, 15, 30, 60, and 90 min after glucose injection using a portable glucose analyzer (Accu-Chek Advantage glucometer, Roche, Mannheim, Germany).
Food consumption was measured at two time points, early (10 weeks) and late (28 weeks) during the 30 weeks of inhalation treatment. The total amount of food was weighed daily in the morning for 5 consecutive days. The daily consumption over the 5 day period was averaged for each mouse to obtain 24-hour food consumption.
Western blot analysis
Western blots were performed on lysates prepared from tissues collected at the end of the experiments. Samples of 30 μg of protein were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA) for 5 min and electrophoresed on 10 or 12.5% SDS-polyacrylamide gels and blotted onto nitrocellulose membrane. Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 2 hours at room temperature and then incubated with primary antibodies overnight at 4°C. Membranes were incubated with either Alex 680 (Molecular Probes) or IR Dye 800 (Rockland, Gilbertsville, PA) secondary antibodies for 1 hour at room temperature. Membranes were visualized using an Odyssey infrared imager (Li-COR, Lincoln, NE) which allows for the simultaneous detection of two proteins. Densitometry analysis was performed using Odyssey software (LI-COR, Lincoln, NE). Antibodies for Western blots were as follows: aldehyde dehydrogenase-1 (ALDH1A1; Abcam, Cambridge, MA), HO-1 (Enzo Life Sciences, Farmingdale, NY), nuclear receptor factor-1 (NRF-1; Rockland, Gilbertsville, PA), peroxisomal proliferating activating receptor-γ coactivator (PGC1-α; Cell Signaling Tech, Boston, MA), uncoupling protein-1 (UCP-1; Sigma, St. Louis, MO) and β-actin (Abcam, Cambridge, MA). All antibodies were used at a ratio of 1:1000 with blocking buffer, the lone exception being β-actin which was used at a ratio of 1:5000. All blots from tissue samples were run in duplicate with 3 samples from each group per gel with prevention reversal samples run on separate gels. These proteins were selected based upon our previous study which demonstrated that these proteins are regulated by CO .
All data are presented as mean ± s.e.m. Differences between treatment groups were determined using one-way analysis of variance with a post hoc test (Dunnett’s). A P < 0.05 was considered to be significant. All analyses were performed with SigmaStat (Systat software Inc., Richmond, CA, USA).