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.

Energy metabolism and expression of uncoupling proteins 1, 2, and 3 after 21 days of recovery from intracerebroventricular mouse leptin in rats.

Animals tend to maintain a lower body weight for an extended period after leptin administration has ended. This may be due to an enhancement of metabolic rate that persists after treatment withdrawal. Our objectives were to determine the period of leptin influence, when injected intracerebroventricularly (icv), on food intake, body weight, and energy expenditure. Additionally, the relationship between expressions of UCP1, UCP2, and UCP3 in different adipose tissues and heat production (HP) was assessed. Twenty-four adult male Sprague-Dawley rats were injected intracerebroventricularly with either 10 g mouse leptin or 10 l vehicle once per day for 4 days. At 24 h after the last injection, one group was killed while the other was placed in calorimetry chambers and monitored for 21 days of recovery. Leptin-injected rats exhibited an overshoot of food intake and respiratory quotient (RQ) during recovery, but body weight remained significantly lower up to 6 days. HP decreased in both groups over time but remained higher in the leptin group through recovery. However, retained energy (RE) was significantly greater than control for about 8 days. Overall, UCP expression was reduced at the end of recovery in parallel with the decline in HP. Brown adipose tissue (BAT) was the most responsive to leptin administration by dramatically changing UCP1 and UCP3 mRNA levels. Our data show that leptin has extended effects on energy expenditure but relieves control on food intake and RQ after treatment withdrawal. This translated into a reduced positive energy balance that slowed body weight recovery.

Inhibition of TNF-alpha induced ICAM-1, VCAM-1 and E-selectin expression by selenium.

The initiation of an atherosclerotic lesion involves an endothelial cell pro-inflammatory state that recruits leukocytes and promotes their movement across the endothelium. These processes require endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin). Tumor necrosis factor-alpha (TNF-alpha) is a powerful inducer of these adhesion molecules. Selenium status is known to affect the rate of atherosclerosis. These experiments tested whether selenium alters cytokine-induced expression of these adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were pretreated for 24 h with sodium selenite (0-2 microM) and then treated with 0 or 50 U/ml TNF-alpha in the presence of 0-2 microM selenite. ICAM-1, VCAM-1 and E-selectin were detected by ELISA and their mRNAs were evaluated by Northern blots. Selenite significantly inhibited TNF-alpha-induced expression of each adhesion molecule in a dose-dependent manner and reduced the level of the respective mRNAs. Nuclear factor-kappa B (NF-kappa B) is required for transcription of these adhesion molecule genes. Western blot analysis revealed that selenite did not inhibit the translocation of the p65 subunit of NF-kappa B to the nucleus. In conclusion, these data indicate selenium can modulate cytokine-induced expression of ICAM-1, VCAM-1 and E-selectin in HUVECs without interfering with translocation of NF-kappa B.