Expression of uncoupling protein‐3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting

Abstract

Uncoupling protein-3 (UCP3) is a mitochondrial innermembrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.

Resistance to high-fat-diet-induced obesity and sexual dimorphism in the metabolic responses of transgenic mice with moderate uncoupling protein 3 overexpression in glycolytic skeletal muscles

“…Protein analysis Mitochondria were prepared from gastrocnemius and soleus muscles as previously described [25]. Electrophoresis of 10 μg of mitochondrial proteins was performed on a 12% SDS-PAGE gel; proteins were then transferred on to nitrocellulose membranes (Hybond ECL; GE Healthcare, Orsay, France).…”

“…Membranes were incubated with an antibody raised against h-UCP3 (Antibody CabrX; Research Diagnostics, San Antonio, TX, USA). The antibody has been validated for detection of both h-UCP3 and mouse UCP3 (m-UCP3) [25]. A single amino acid is different between human and mice in the 14 amino acid peptide used to raise the antibody.…”

Peripheral Administration of the Melanocortin-4 Receptor Antagonist NBI-12i Ameliorates Uremia-Associated Cachexia in Mice

“…9,10 We tested whether NBI-12i has additional metabolic advantages beyond the nutritional effects of stimulating appetite by measuring the basal metabolic rate of individual mice. Basal metabolic rate was increased in N mice (4182 Ϯ 48 ml/kg per h) compared with S mice (3930 Ϯ 24 ml/kg per h; P Ͻ 0.007; Figure 3A).…”

“…BAT mitochondrial protein was extracted according to Jimenez et al 9 Protein concentration was determined using Bio-Rad Dc Assay. Equal amount of total protein (approximately 3 g) was analyzed on a 10% SDS-PAGE gel in reduced condition and electrotransferred to a nitrocellulose paper.…”

Control of 4E‐BP1 expression in mouse brown adipose tissue by the β3‐adrenoceptor

“…Thirty micrograms of supernatant was dried under vacuum and resuspended in 10 ll of a loading buffer containing 20% glycerol, 6% SDS and 0.02% bromophenol blue in 0.25 M Tris/HCl, pH 6.8. Western blots were performed as previously described [14] using rabbit polyclonal antibody #11211, which detects mouse 4E-BP1 and 4E-BP2…”