Low content of mitochondrial ATPase in brown adipose tissue is the result of post‐transcriptional regulation

Abstract

The mI~NA levels of ATPase/5, ATPase 6, cytochrome oxidase (COX) Vlb and COX I subunit~ were found to be 2.4~13.8-fold higher in brown adipose tissue (BAT) than in heart, skeletal muscle, brain and liver of mice. The comparison with tissue contents of ATPase and COX revealed that the sele~=tive, 5-1 l-fold reduction of ATPase in BAT is not caused by decreased transcription of ATPase genes. Likewise, the ATPase ]~ and COX Vlb mRNA levels in cultured brown adipocytes were also not influenced by norepinephrine, which activated the expression of the UCP gene by two orders of magnitude. The results indicate that the biosynthesis of mitochondrial ATPase in BAT is post-transcriptionally regulatm~l. lional events are responsible for the selective reduction of the ATPase content in BAT. MATERIALS AND METHODSFour-week-old mice of Balb/c outbred strain were maintained at room temperature, on a 12 h light/12 h dark cycle. When indicated, animals were exposed to 4°C prior to use. The animals were killed by decapitation and ti.ssue samples for RNA isolation were frozen in liquid nitrogen. For other assays, 5~ homog~.:ates [w/v) were prepared in 0.25 M sucrose, 10 mM Tris/HCI, I mM EDTA, pH 7.4, and filtered through a 250/Jm nylon screen.The stromal-vascular ceils from BAT were isolated and cultivated as previously [71. Experiments were performed with cells cultured for 7 days.Total RNA was isolated as described in [8] and checked for purity and integrity. For Northern blot analysis, 20Mg RNA aliqnots were denatured in the presence of glyoxaL separated by eleetrophoresis in 1.2% agaros¢ gel and transferred to a nylon membrane (Hybond-N, Amersham) according to standard procedures [9]. Hybridization was carried out overnight at 68°C in 5x Dcnhardt's solution, 5x SSC, 50 mM sodium phosphate, pH 6.5. 0. !% SDS and 250 pg/ml of herring sperm DNA. Blots were washed in 2× SSC, 0.1 ~b SDS (2x |5 min, room temp.) and 0.Ix SSC, 0.1% SDS (2× 15 rain, 55°C). Following autoradiography the blots were stripped for 60 rain at 95~C in 0. I SDS and sequentially re-hybridized with several DNA pr3bes.The eDNA probes were generously provided by the following investigators: 1.26 kb E.coR! fragment of the rat ATPase /~-subunR eDNA in pUCI9

Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c‐Fo subunit P1 isoform

“…Physiologically, the low amount of the ATP synthase is compatible with the fact that the energy from oxidative processes is released as heat, not through a futile ATP turnover cycle but through the activity of the brown adipose tissue‐specific uncoupling protein UCP1 (7). Strikingly, however, the majority of the ATP synthase subunit genes are expressed coordinately with other OXPHOS enzymes, and the mRNA levels for most subunits are thus very high compared to that in most other tissues (10, 11). The remarkable issue is thus the means by which the very low levels of ATP synthase are achieved in brown adipose tissue.…”

“…Brown adipose tissue (BAT) (7), the thermogenic organ of mammals, contains extremely high amounts of the mitochondrial respiratory chain enzymes but remarkably low amounts of the F 1 F o ‐ATPase (8, 9). Surprisingly, in contrast to this low protein content, at the mRNA level the majority of the F 1 F o ‐ATPase subunits are present in abundant amounts (10). The only subunit with remarkably low levels of mRNA in this tissue is the c‐F o subunit (11).…”

Expression of interleukin‐1α and interleukin‐1 receptor type I genes in murine brown adipose tissue

“…Northern blots (20,ug total RNA aliquots) and sequentional hybridization with several probes were performed essentially as before [17].…”

Gender-related differences in morphology and thermogenic capacity of brown adipose tissue mitochondrial subpopulations

“…One of the main characteristics of the BAT mitochondria is a strong oxidative potential accompanied by a low phosphorylative capacity (Cannon and Vogel, 1977;Houstek et al, 1991;Lindberg et al, 1967), which is reflected when a quotient between COX and ATP synthase activity is calculated. Theoretically, a greater COX/ATP synthase ratio would be related to a greater UCP1 content.…”