(A) eNOS mRNA levels, measured by means of quantitative RT-PCR in WAT of genetically obese mice (ob/ob) and rats (fa/fa) and of environmentally obese mice (DIO) compared to respective controls (+/+, wild-type; SC, standard chow–fed animals). The cycle number at which the various transcripts were detectable was compared with that of β-actin as an internal control and expressed as arbitrary units versus values in control animals taken as 1.0 (n = 5 experiments). ***P < 0.01. (B) eNOS protein was detected by immunoblot analysis (1 experiment representative of 5 reproducible ones) in WAT of ob/ob mice, fa/fa rats, and DIO mice compared to respective controls. Numbers indicate the relative values from the densitometric analysis (normalized to β-actin) relative to controls, which were assigned a value of 1.0.

WAT from control or ob/ob mice was processed to perform stereological analysis of mitochondria. The tissues were fixed and sectioned randomly and prepared for electron microscopy. Representative electron micrographs are shown at left. M, mitochondria; L, lipid droplets. At right, 50 photographs at a magnification of ×11,500 were taken, and the area and density of 998 wild-type and 1,112 ob/ob mitochondria were determined. Results are mean ± SEM (n = 3 per group). ***P < 0.01, *P < 0.05.

(A) eNOS mRNA analyzed by quantitative RT-PCR in WAT of standard chow–fed mice (control), DIO mice, or DIO p55^–/– mice. (B) Oxygen consumption and (C) ATP levels in WAT of DIO and DIO p55^–/– mice were compared with those of standard chow–fed mice. Relative values are mean ± SEM (n = 3 per group) when control measurements were assigned a value of 1.0. ***P < 0.01, *P < 0.05 versus standard chow; ^†P < 0.05 versus DIO.

(A) PGC-1α, NRF-1, and Tfam mRNA, analyzed by means of quantitative RT-PCR with gene-specific oligonucleotide probes in WAT of obese and control animals. The cycle number at which the various transcripts were detectable was compared to that of β-actin as an internal control, and expressed as arbitrary units versus values in control animals taken as 1.0 (n = 5 experiments). Shown at top is mtDNA analysis; 1 experiment representative of 5 gels (n = 5 per group). Numbers indicate relative amounts from the densitometric analysis when control measurements were assigned a value of 1.0. Leftmost lanes show DNA markers (–). (B) COX IV and Cyt c proteins were detected by immunoblot analysis (1 experiment representative of 5 reproducible ones) in WAT of obese and control animals. Numbers indicate relative values from the densitometric analysis (normalized to β-actin) relative to controls, which were assigned a value of 1.0. (C) Oxygen consumption by WAT of obese and control animals was measured in a gas-tight chamber using an O₂ electrode. Oxygen consumption values were normalized to the tissue protein content (n = 3 experiments). ***P < 0.01, *P < 0.05.

(A) eNOS mRNA and protein levels were analyzed by quantitative RT-PCR or immunoblotting, respectively, in WAT of wild-type, ob/ob, ob/ob p55^–/–, ob/ob p75^–/–, and ob/ob p55^–/–p75^–/– mice. Relative values are mean ± SEM (n = 3 per group) when control measurements were assigned a value of 1.0. (B) PGC-1α, NRF-1, and Tfam mRNA were analyzed by means of quantitative RT-PCR with gene-specific oligonucleotide probes in WAT of wild-type, ob/ob, ob/ob p55^–/–, ob/ob p75^–/–, and ob/ob p55^–/–p75^–/– mice. The cycle number at which the various transcripts were detectable was compared with that of β-actin as an internal control and expressed as arbitrary units. (C) COX IV and Cyt c protein levels were detected by densitometric analysis of immunoblots in WAT of wild-type, ob/ob, ob/ob p55^–/–, ob/ob p75^–/–, and ob/ob p55^–/–p75^–/– mice. ***P < 0.01, *P < 0.05 versus wild-type (n = 3 per group); ^†P < 0.05 versus ob/ob.

(A) eNOS mRNA and protein were analyzed by quantitative RT-PCR and immunoblotting, respectively, in untreated cells (C, control) or after exposure to TNF-α at different doses for 2 days. (B) PGC-1α, NRF-1, and Tfam mRNA levels were determined in untreated WAT cells and after treatment with 1 nM TNF-α alone or in combination with 50 μM DETA-NO for 2 days. (C) COX IV and Cyt c protein levels and (D) oxygen consumption were measured in untreated WAT cells and after treatment with 1 nM (C) or different doses (D) of TNF-α alone or in combination with 50 μM DETA-NO for 2 days. Relative values are mean ± SEM (n = 4 different experiments) when control measurements were assigned a value of 1.0. **P < 0.01, *P < 0.05 versus untreated cells.