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The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.

Cold acclimation induced an increase of spin-lattice relaxation time (T1) of proton nuclear magnetic resonance in rat brown adipose tissue with concomitant increase of water content, while heat acclimation did not effect T1 of this tissue, although its water content was significantly decreased. Temperature acclimation did not affect either T1 or water content of neck muscle.

A mutation in the BALB/cHeA subline of mice has resulted in loss of the glycerol-3-phosphate dehydrogenase (glycerol-P dehydrogenase) activity in adult tissues. Analysis of F2 offspring segregating for enzyme activity levels and a PvuII restriction fragment length polymorphism in the Gdc-1 gene on Chromosome 15 indicates that the mutation has occurred in or around the structural gene. Southern blot analysis failed to detect any major change due to chromosomal insertion, deletion, or rearrangement of the mutant gene. In vitro measurements of transcription in isolated nuclei failed to detect any difference in transcription between the BALB/cHeA and BALB/cBy strains of mice. However, Northern blot analysis with poly(A+) RNA isolated from brown fat shows that the mutant has about a 50-fold reduction in hybridizable transcripts of altered size. The data suggest that the mutation affected the processing of nuclear transcripts of glycerol-P dehydrogenase. The morphological, physiological, and reproductive characteristics of the mutant mice appear normal. This suggests that alternate pathways of energy metabolism and lipid synthesis exist which obviate the function of glycerol-P dehydrogenase.

The effects of both exercise training and intermittent cold acclimation on heat production (shivering and nonshivering thermogenesis (NST)) in rats were studied. Warm-acclimated rats (housed at 24 degrees C, WA) and intermittently cold-acclimated rats (exposed daily to -5 degrees C for 2 h, CA) were forced to run (25 m.min-1 for 1 h) every day (WA-T and CA-T). WA and CA left sedentary (WA-S and CA-S) served as controls. Norepinephrine (NE)-induced thermogenic capacity assessed from the increment of oxygen consumption (VO2) and colonic temperature (Tc) were measured 4 weeks after commencing acclimation and exercise training. The thermogenic capacity was greater in CA than in WA. However, in WA, WA-T responded to NE less than WA-S, whereas the response of CA-T and CA-S did not differ. Wet weight of interscapular brown adipose tissue (IBAT) and its protein (and dry matter--regarded to be highly representative of protein) content were larger in CA than in WA. Respective sedentary and exercised groups of rats had similar IBAT protein (and dry matter) content although tissue weight was lighter in WA-T than in WA-S. Lipid content of IBAT was also larger in CA than in WA. IBAT of WA-T had less lipid compared to that of WA-S while no difference was seen between CA-S and CA-T. Shivering activity during acute cold (4 degrees C) exposure was less in CA compared to WA and there was no difference between respective groups of exercised and sedentary rats. Propranolol, a blocker of NE-dependent NST, eliminated the difference in shivering among these four groups. When exposed to severe cold (-10 or -20 degrees C), the fall in Tc of rats fasted for 18 h was greater in WA than in CA. CA-T showed a greater decrease in Tc than CA-S during -20 degrees C exposure while it did not differ during -10 degrees C exposure. On the other hand, Tc of WA-T and WA-S did not differ significantly during either cold exposure period. These results suggest that exercise training in rats housed at 24 degrees C suppresses NE-dependent NST whereas another nonshivering thermogenic mechanism (NE-independent) may compensate this suppression. However, NE-dependent NST of WA-S and WA-T did not parallel protein (dry matter) content of IBAT; no difference existed in IBAT protein between these two groups.(ABSTRACT TRUNCATED AT 400 WORDS)

The presence of angiotensinogen messenger RNA (mRNA) was detected in rat vascular and adipose tissue. Angiotensinogen mRNA in rat aorta was localized in the adventitia and surrounding adipose tissue, and not in the vascular smooth muscle. Freshly dispersed and cultured endothelial and aortic smooth muscle cells did not contain detectable amounts of angiotensinogen mRNA. In addition to periaortic adipose tissue, angiotensinogen mRNA was present in other fat depots of both brown and white types. To examine regulation of angiotensinogen gene expression, Sprague-Dawley rats were treated with angiotensin converting enzyme inhibitor or underwent bilateral nephrectomy. Relative levels of angiotensinogen mRNA in brown adipose tissues increased dramatically by 48 hours after bilateral nephrectomy. However, only one source of brown adipose tissue showed increased angiotensinogen mRNA levels after animals were treated for 5 days with converting enzyme inhibitor. In addition, angiotensinogen was released into the medium from incubated adipose tissues with levels increasing over a 2-hour period. These results demonstrate that angiotensinogen is synthesized by adipose tissue in the rat and may play a role in the function of this tissue.

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein.

In order to ascertain possible involvement of polyamines in the physiological regulation of brown fat function, effect of temperature acclimation on the polyamine contents of this tissue and effects of polyamines on the noradrenaline-induced thermogenesis is isolated brown adipocytes were investigated in rats. Daily urinary excretion of polyamines measured collectively for spermidine and spermidine per body weight was decreased in heat acclimation and increased in cold acclimation. Polyamine concentrations per fresh weight of brown fat showed extremely low values compared with those of other tissues previously reported. Putrescine and spermidine contents per fat-free dry matter of brown fat were decreased in cold acclimation, but were not affected in heat acclimation. Spermidine and spermine inhibited the noradrenaline-induced thermogenesis of brown adipocytes dose-dependently. These results suggest that polyamines regulate the heat production of brown adipose tissue in an inhibitory way and cold acclimation potentiates heat production of this tissue by reducing polyamine levels.

1. The rates of oxygen consumption and the colonic temperatures were monitored from birth in rabbits delivered by Caesarean section at term. The rates of oxygen consumption at 25 and 36 degrees C rose over the first 12 hr of life.2. Thirty rabbits were kept unfed in an environment of 25 degrees C from birth. Extra oxygen consumption, in response to cold exposure, failed 24-34 hr later. Twenty-four of these rabbits were allowed to recover in an environment of 36 degrees C. The metabolic response to cold exposure did not return in those kept unfed or given glucose but those fed with rabbit's milk showed significant recovery when re-exposed to cold 4 or 18 hr later.3. The tissue glyceride content of brown and white adipose tissue and the liver were greatly reduced once the animals failed to respond to cold exposure and no significant rise was detected during the recovery period after a milk feed.4. Another twelve rabbits were kept unfed in an environment of 36 degrees C. These animals showed a poor metabolic response to cold exposure after 48 hr, although their brown adipose tissue was still replete with fat and the blood glucose rose to normal values during cold exposure.5. These results suggest that an increase in oxygen consumption in response to cold exposure can occur even though brown adipose tissue is largely depleted of glyceride provided that an alternative supply of fuel is available. Glucose alone is not an adequate source of energy.

The total nitrogen and L-hydroxyproline concentrations of two intra-abdominal and two subcutaneous sites of adipose tissue were measured in male and female Wistar rats. At 100 g body weight the nitrogen concentration of intra-abdominal adipose tissue was slightly lower than that of subcutaneous adipose tissue. As age advanced beyond sexual maturity, the nitrogen content of intra-abdominal adipose tissue was steadily reduced, whereas that of subcutaneous tissue did not change. The L-hydroxyproline concentration of intra-abdominal adipose tissue was also lower than in subcutaneous adipose tissue. In male rats it rose in most adipose tissue sites when sexual maturity was reached, then fell with advancing age. In female rats the pattern of change was less consistent, but the l-hydroxyproline content of intra-abdominal adipose tissue was reduced as age increased.

1. The rates of oxygen consumption were measured in 6-8-day-old rabbits at 34 and 15 degrees C after varying periods of starvation and cold exposure. At the start of the experiment the rabbits had been fasted for 24 hr. Eight rabbits were studied immediately, six after 24 and six after 48 hr in a cold environment (20 degrees C), and twelve after a further 48 hr in a warm environment (34 degrees C). All the animals had a similar increase in oxygen consumption during the final hour of cold exposure (15 degrees C).2. The rabbits kept at 20 degrees C lost 83% of the fat stored in their brown adipose tissue within 24 hr and a further 11% in the next 24 hr. The fat content of white adipose tissue had fallen by 75% at 48 hr. In contrast rabbits kept unfed at 34 degrees C had lost 47% of the fat in brown adipose tissue and 44% of the fat in white adipose tissue after 48 hr.3. In six rabbits subcutaneous thermocouples demonstrated that local heat production continued in brown adipose tissue after 48 hr cold exposure.4. In the rabbits kept at 34 degrees C the final cold exposure caused a large increase in the serum free fatty acid and glycerol concentrations. Much lower concentrations were found in rabbits kept at 20 degrees C.5. The results show that the fat stored in the brown adipose tissue of young rabbits exposed to cold is preferentially used for heat production. When this store of fat is exhausted, brown adipose tissue still produces heat presumably by oxidizing fat and glucose taken from the circulation.

Brown adipose tissue of the rat has been found to have an unusually high activity of mitohondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) when assayed both by a histochemical staining procedure and by a quantitative biochemical method with isolated mitochondria. In contrast to succinic, glutamic, and beta-hydroxybutyrate dehydrogenases, all mitochondrial enzymes, the activity of alpha-GPD in brown fat was 10 times that in liver, more than 20 times that in white adipose tissue, and 9 times that in kidney. The soluble NAD-linked alpha-GPD was also higher in brown adipose tissue than in white adipose tissue, liver, or kidney, but the differences were much less marked. The possible importance of the high activity of mitochondrial alpha-GPD in the regulation of synthesis of esterified lipid and in thermogenesis in brown fat is discussed.