Contributions of studies on uncoupling proteins to research on metabolic diseases.

The coupling of O2 consumption to ADP phosphorylation in mitochondria is partial. This is particularly obvious in brown adipocyte mitochondria which use a regulated uncoupling mechanism generating heat production from substrate oxidation, and catalysing thermogenesis in rodents or infants in response to cold, and arousing hibernators. In the case of brown adipose tissue, the uncoupling mechanism is related to a specific protein in the inner mitochondrial membrane referred to as UCP1.

Leptin and corticosterone have opposite effects on food intake and the expression of UCP1 mRNA in brown adipose tissue of lep(ob)/lep(ob) mice.

The present study was conducted to assess the interaction effect of leptin and corticosterone on food intake and the expression of uncoupling protein 1 (UCP1) mRNA in interscapular brown adipose tissue (IBAT). To this end, a 3 x 3 factorial experiment was designed in which adrenalectomized (ADX) lep(ob)/lep(ob) mice were subjected to three doses of corticosterone and three doses of leptin. The results confirm the anorectic and orexigenic effects of leptin and corticosterone, respectively.

Distribution of the uncoupling protein 2 mRNA in the mouse brain.

The present study was conducted to investigate the brain distribution of the recently cloned uncoupling protein 2 (UCP2). Northern blot analyses were first carried out to confirm the presence of UCP2 in the brain. These analyses revealed the brain presence of UCP2 mRNA and the absence of the mRNAs encoding uncoupling protein 1 and uncoupling protein 3.

Up-regulation of UCP-2 gene expression by PPAR agonists in preadipose and adipose cells.

UCP-2 is a member of the emerging family of UCP homologues. Upon high-fat feeding, UCP-2 mRNA levels are increased in epididymal fat pads of A/J mice, suggesting that the flux of fatty acids entering adipose tissue may regulate UCP-2 gene expression. Since fatty acids act as positive transcriptional regulators of lipid-related genes by means of peroxisome proliferator-activated receptors (PPARs), the regulation of UCP-2 gene expression by PPAR agonists (carbacyclin, alpha-bromopalmitate, BRL49653) has been examined in mouse preadipose and adipose cells in primary cultures or from clonal lines (Ob1771, 3T3-F442A, 1B8).

Leptin gene is expressed in rat brown adipose tissue at birth.

The ob gene product leptin is secreted from adipose tissue. Leptin has dramatic effects on food intake and energy expenditure in rodents. Brown adipose tissue is the first form of adipose tissue to appear during development, and is present at birth in most species.

Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.

A mitochondrial protein called uncoupling protein (UCP1) plays an important role in generating heat and burning calories by creating a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane in brown adipose tissue, without coupling to any other energy-consuming process. This pathway has been implicated in the regulation of body temperature, body composition and glucose metabolism. However, UCP1-containing brown adipose tissue is unlikely to be involved in weight regulation in adult large-size animals and humans living in a thermoneutral environment (one where an animal does not have to increase oxygen consumption or energy expenditure to lose or gain heat to maintain body temperature), as there is little brown adipose tissue present.

Essential cis-acting elements in rat uncoupling protein gene are in an enhancer containing a complex retinoic acid response domain.

Transgenic mice were generated with a transgene containing the 211-base pair (bp) enhancer and 0.4 kilobase pairs of 5'-flanking DNA of the uncoupling protein (ucp) gene. Expression of this transgene was restricted to brown adipose tissue and was inducible by cold exposure or treatment of transgenic mice by norepinephrine, retinoic acid (RA), or CL-316,243 beta3-adrenoreceptor agonist.

Evidence from in vitro differentiating cells that adrenoceptor agonists can increase uncoupling protein mRNA level in adipocytes of adult humans: an RT-PCR study.

In vivo data have suggested that adrenergic signals can reactivate dormant brown adipocytes in adult humans. We report here a system based on primary cultures of perirenal adipocytes from human adults and reverse transcription-PCR of uncoupling protein mRNA. Norepinephrine and compounds classified as beta 3-adrenoceptor agonists in rodents increased uncoupling protein mRNA level in human adipocytes (presumably brown adipocytes).

In vitro interactions between nuclear proteins and uncoupling protein gene promoter reveal several putative transactivating factors including Ets1, retinoid X receptor, thyroid hormone receptor, and a CACCC box-binding protein.

Previous studies of rat ucp (uncoupling protein) gene organization carried out in this laboratory identified regulatory sequences located in the 5'-flanking region. In this work, DNase I footprint analysis of the enhancer revealed two domains at base pairs (bp) -2444 to -2423 and bp -2352 to -2319. The former domain can bind in vitro, in a cooperative manner, factors related to nuclear factor 1 and Ets1; the latter domain contains a type 3 directly repeated sequence that was shown to be able to bind the retinoid X and triiodothyronine receptors.

Characterization of the novel brown adipocyte cell line HIB 1B. Adrenergic pathways involved in regulation of uncoupling protein gene expression.

The HIB 1B cell line, derived from a brown fat tumor of a transgenic mouse, is the first established brown adipocyte cell line capable of expressing the brown fat-specific mitochondrial uncoupling protein (UCP). UCP gene expression, which was virtually undetectable under basic conditions, was stimulated by acute catecholamine or cyclic AMP treatment to levels comparable to primary cultures of brown adipocytes. Elevation of UCP mRNA levels following stimulation was very rapid but transient, decreasing after about 4 hours with a half-life between 9 and 13 hours.

The beta 3-adrenoceptor agonist ICI-D7114 is not as efficient on reinduction of uncoupling protein mRNA in sheep as it is in dogs and smaller species.

Adipose tissue in newborn lambs is brown, but within a few days it is transformed into white adipose tissue. In the same way, preadipocytes cultured in serum-free chemically defined medium achieve full differentiation and express uncoupling protein (UCP), a marker of brown adipose tissue, when isolated from perirenal adipose tissue of the newborn, whereas they no longer express UCP when isolated from older lambs. The effects of a chronic stimulation of adipose tissue by novel beta 3-adrenoceptor agonist (ICI D7114) on the maintenance after birth and on the reinduction in older lambs of UCP mRNA in adipose tissue were studied.

Regulation of UCP gene expression in brown adipocytes differentiated in primary culture. Effects of a new beta-adrenoceptor agonist.

Primary cultures of precursor cells from mouse and rat brown adipose tissue (BAT) were used to study the effect of a new beta-agonist (ICI D7114) on the uncoupling protein (UCP) gene expression. ICI 215001 (the active metabolite of D7114) increased the expression of UCP and its mRNA in brown adipocytes differentiating in vitro in a dose-dependent manner. This stimulating effect was not inhibited by propranolol, a non-specific beta-antagonist, but was partially reduced by bupranolol, a beta 3-antagonist.

Comment to Shinohara et al. (1991) FEBS Letters 293, 173-174. The uncoupling protein is not expressed in rat liver.

Using Northern blot analysis, immunoblotting with purified antibodies and Polymerase Chain Reaction analysis, we were unable to detect the Uncoupling Protein-UCP or its mRNA in liver of control, cold-exposed or newborn rats. The unique expression of this protein in brown adipocytes was confirmed. These data refute the surprising recent report on UCP expression in rat liver (Shinohara (1991) FEBS Lett.

Beta 3-adrenergic receptor stimulation restores message and expression of brown-fat mitochondrial uncoupling protein in adult dogs.

Brown adipose tissue (BAT) is present throughout life in rodents and plays an important role in energy balance. However, whereas BAT is clearly recognizable in the neonates of larger mammals (including dogs, cats, sheep, cattle, and humans), it is undetectable or present in only small quantities in adults of these species and is replaced by a tissue with the gross characteristics of white adipose tissue. Here we provide evidence that treatment of adult dogs with a beta 3-adrenergic receptor agonist (ICI D7114) that has thermogenic and antiobesity properties leads to the appearance of BAT at several anatomical sites.

Effects of fasting and refeeding on the level of uncoupling protein mRNA in rat brown adipose tissue: evidence for diet-induced and cold-induced responses.

Brown adipose tissue (BAT) is characterized by the existence of a unique mitochondrial protein (uncoupling protein or UCP) that uncouples oxidative phosphorylation and thus allows heat production. Its role in thermogenesis has been emphasized in recent years in response to cold stress (nonshivering thermogenesis, NST) as well as to hyperphagia (diet-induced thermogenesis, DIT). The present work was a first attempt to determine whether varying nutritional conditions could affect UCP gene expression.

Increased nonshivering thermogenesis, brown fat cytochrome-c oxidase activity, GDP binding, and uncoupling protein mRNA levels after short daily cold exposure of Phodopus sungorus.

In their natural environment, burrowing rodents experience rather fluctuating ambient temperatures and are acutely cold exposed only for short periods outside their burrows. The effect of short daily cold exposure on basal metabolic rate, nonshivering thermogenesis, brown fat thermogenesis, and uncoupling protein mRNA was studied in the Djungarian hamster, Phodopus sungorus. They were kept at 23 degrees C and exposed to 5 degrees C daily either for one 4-h period or twice for 2 h (in 12-h intervals).

Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species. Sudden occurrence of uncoupling protein mRNA during embryogenesis and its disappearance after birth.

Samples of adipose tissue were obtained from different sites in bovine and ovine foetuses and newborns. RNA was isolated and analysed using bovine cDNA and ovine genomic probe for uncoupling protein (UCP), cDNA for subunits III and IV of cytochrome c oxidase and cDNA for ADP/ATP carrier. UCP mRNA was characterized for the first time in foetal bovine and ovine adipose tissue.

Alanine turnover rate and its hepatic metabolism are increased in midpregnant rat.

At mid pregnancy (12th day) fed rats exhibit a drop of blood alanine level when the weight of the fetoplacental unit is still negligible. A primed infusion A-V method was used to measure alanine kinetics in fed midpregnant rats and virgin controls, using L-[2,3-3H]alanine as a tracer. Alanine turnover and metabolic clearance rates were higher on day 12 than in virgin controls.

Lipoprotein lipase activity in skeletal muscle and brown adipose tissue of pregnant and lactating rats.

Changes in lipoprotein lipase (LPL) activity during pregnancy and lactation were followed in skeletal muscles and interscapular brown adipose tissue (BAT) of rats fed two diets differing in energy density (high carbohydrate or high fat). Rats were decapitated after 7, 19 or 21 d of pregnancy or after 3 or 12 d of lactation. Virgin rats and females separated from their litter just after delivery were used as nonpregnant and nonlactating controls, respectively.

Urea concentration and ornithine decarboxylase in liver of female rats.

In virgin female rats thioacetamide administration (1 mg/100 g body wt) induced a 16-fold increase in liver ornithine decarboxylase (ODC) activity and a significant decrease (19%) in hepatic urea concentration. The ornithine-metabolizing enzymes, ornithine-oxo-acid aminotransferase and ornithine carbamoyltransferase, were not modified by the treatment; only carbamoyltransferase, were not modified by the treatment; only carbamoyl-phosphate synthetase I activity was significantly reduced. In 19-day pregnant rats DL-alpha-difluoromethylornithine treatment inhibited the expression of enhanced ODC activity occurring normally at this stage of pregnancy.

[Effect of a hyperlipidic diet on lipogenesis, storage and utilization of lipids in the pregnant rat].

The aim of the present investigation was to determine the amount of fat accumulated and mobilized during pregnancy in rats fed either an hyperglucidic or an hyperlipidic diet. The feeding of an hyperlipidic diet increased the liver lipid content and did not modify the composition of the mammary gland. Fat was deposited till day 19 of pregnancy; after that date the fat was mobilized.

Liver ornithine decarboxylase in pregnant rats fed two levels of casein.

Liver ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) activities were assessed at 2200 h (prandial phase) and at 1000 h (postprandial phase) in virgin and in pregnant (day 13-20) rats fed on different levels of casein and carbohydrate. In virgin rats, ODC levels were higher at 2200 h after resumption of eating than at 1000 hours, the inductive effect being greater with the high-casein than with the low-casein diet. Rapid deinduction followed termination of eating, resulting in equally low enzyme levels at 1000 h with both diets.

Circadian feeding pattern in pregnant rats fed three levels of protein.

Food intake was measured at regular intervals over 24 h in pregnant and non-pregnant female rats fed diets of different protein content: 10, 16 and 32%. During the course of pregnancy, a first period of hyperphagia was observed (days 2-12) irrespective of the composition of the diet. A second phase of hyperphagia occurred later (days 16-19) which was more marked with the better balanced diet (16% protein).

Possible metabolic implications of pyruvate and lactate accumulation in the liver of pregnant rats.

Experiments were designed to investigate whether the metabolic responses of pregnant females are in keeping with the known state of gestational hyperinsulinemia. Groups of female rats fed a 32% protein diet were killed on days 13, 15, 17, 19 and 21 of pregnancy, during either daytime or during night-time. Liver pyruvate kinase and glucose-6-phosphate dehydrogenase activities were increased over nonpregnant values from day 13 onward in agreement with what can be expected as a result of the gestational hyperinsulinemia.

[Effects of the combination of a pesticide, lindane, and a sulfur depleted diet on pregnancy in the rat].

Effects of assocation of lindane ingestion and sulfur depletion on pregnancy in the rat. Sulfoconjugation stands as one of the main processes of foreign compound detoxication in mammals. When feeding conditions are adequate, sulfate ions necessary to form sulfoconjugates are provided by the sulfur aminoacids of the diet.