Teresa Mampel

Thermogenic Activation Induces FGF21 Expression and Release in Brown Adipose Tissue

FGF21 is a novel metabolic regulator involved in the control of glucose homeostasis, insulin sensitivity, and ketogenesis. The liver has been considered the main site of production and release of FGF21 into the blood. Here, we show that, after thermogenic activation, brown adipose tissue becomes a source of systemic FGF21. This is due to a powerful cAMP-mediated pathway of regulation of FGF21 gene transcription. Norepinephrine, acting via β-adrenergic, cAMP-mediated, mechanisms and subsequent activation of protein kinase A and p38 MAPK, induces FGF21 gene transcription and also FGF21 release in brown adipocytes. ATF2 binding to the FGF21 gene promoter mediates cAMP-dependent induction of FGF21 gene transcription. FGF21 release by brown fat in vivo was assessed directly by analyzing arteriovenous differences in FGF21 concentration across interscapular brown fat, in combination with blood flow to brown adipose tissue and assessment of FGF21 half-life. This analysis demonstrates that exposure of rats to cold induced a marked release of FGF21 by brown fat in vivo, in association with a reduction in systemic FGF21 half-life. The present findings lead to the recognition of a novel pathway of regulation the FGF21 gene and an endocrine role of brown fat, as a source of FGF21 that may be especially relevant in conditions of activation of thermogenic activity.

Adenine nucleotide translocase 3 (ANT3) overexpression induces apoptosis in cultured cells

Mitochondrial adenine nucleotide translocase 1 (ANT1), but not ANT2, can dominantly induce apoptosis [Bauer et al. (1999) J. Cell Biol. 439, 258–262]. Nothing is known, however, about the apoptotic activity of ANT3. We have transfected HeLa cells with the three human ANT isoforms to compare their potential as inducers of apoptosis. Transient overexpression of ANT3 resulted, like ANT1, in apoptosis as shown by an increase in the sub‐G1 fraction, annexin V staining, low ΔΨ m, and activation of caspases 9 and 3. Moreover, the apoptosis produced by ANT3 was inhibited by bongkrekic acid and by cyclosporin A. The pro‐apoptotic activities of the ANT1 and ANT3 isoforms contrast with the lack of apoptotic activity of ANT2. This finding may help to identify the specific factors associated with the pro‐apoptotic activities of ANT isoforms.

Opposite regulation of PPAR-α and -γ gene expression by both their ligands and retinoic acid in brown adipocytes

Abstract The peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors involved in the regulation of lipid metabolism and adipocyte differentiation. Little is known, however, about the control of the expression of the genes encoding each of all three receptor subtypes: α, δ, and γ. We have addressed this question in the brown adipocyte, the only cell type that co-expresses high levels of the three PPAR subtypes. Differentiation of brown adipocytes is associated with enhanced expression of PPAR genes. However, whereas PPARγ and PPARδ genes are already expressed in preadipocytes, the mRNA for PPARα appears suddenly in association with the acquisition of the terminally differentiated phenotype. Both retinoic acid isomers and PPAR agonists, specific for either PPARα or PPARγ, regulate expression of each PPAR subtype gene in the opposite way: they up-regulate PPARα and down-regulate PPARγ. The effects on PPARα mRNA are independent of protein synthesis, whereas inhibition of PPARγ mRNA expression depends on protein synthesis, except when its specific ligand prostaglandin J 2 is used. Our results indicate a strictly opposite autoregulation of PPAR subtypes, which supports specific physiological roles for them in controlling brown fat differentiation and thermogenic activity.

Uncoupling protein‐3 gene expression in skeletal muscle during development is regulated by nutritional factors that alter circulating non‐esterified fatty acids

Uncoupling protein‐3 gene expression in skeletal muscle is up‐regulated during postnatal development of mice. A high‐carbohydrate diet at weaning induces a decrease in uncoupling protein‐3 mRNA levels that does not occur when mice were weaned onto a high‐fat diet. Uncoupling protein‐3 mRNA levels do not increase in response to fasting in young pups. Only after day 15 of life, when fasting increases serum non‐esterified fatty acids, uncoupling protein‐3 mRNA is up‐regulated by starvation. Over‐nutrition or under‐nutrition during lactation increases or decreases, respectively, uncoupling protein‐3 mRNA expression in skeletal muscle. Regulation of uncoupling protein‐3 gene expression in skeletal muscle during development is mediated by ontogenic and nutritional factors determining changes in circulating non‐esterified fatty acids.

Both retinoic-acid-receptor- and retinoid-X-receptor-dependent signalling pathways mediate the induction of the brown-adipose-tissue-uncoupling-protein-1 gene by retinoids.

The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte.

Sequential changes in brown adipose tissue composition, cytochrome oxidase activity and GDP binding throughout pregnancy and lactation in the rat

The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and GDP binding to brown fat mitochondria were unchanged. In early lactation (day 5) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and 15), the amount of tissue and its mitochondrial protein content were minimal and the GDP binding per mitochondrial protein decreased substantially. Scatchard analysis in day-15-lactating rats indicated a large decrease in GDP binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity.

9-cis Retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes

The expression of uncoupling protein‐2 (UCP2) mRNA is up‐regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down‐regulated. 9‐cis Retinoic acid causes a dose‐dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all‐trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9‐cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.

Glucose tolerance and insulin response in offspring of ethanol-treated pregnant rats

The effects of maternal alcohol ingestion on oral glucose tolerance and insulin response were studied just after birth and in 3-day old offspring of rats given ethanol for drinking (25% w/v) during pregnancy. Offspring litter size, litter survival and body weight were reduced as a consequence of maternal alcohol treatment. Basal plasma insulin levels were augmented in pups from alcoholized mothers just after birth, despite the fact that blood glucose did not change. Maternal alcohol consumption caused glucose intolerance associated with unchanged insulin response in pups just after birth whereas 3-day old pups from alcoholized mothers showed normal glucose tolerance associated with increased insulin response. Data indicate that chronic maternal ethanol treatment may cause impaired insulin sensitivity in the offspring.

Lipoprotein lipase mRNA expression in brown adipose tissue: translational and/or posttranslational events are involved in the modulation of enzyme activity.

Lipoprotein lipase mRNA abundance in rat brown adipose tissue increases during the first 24 h of cold exposure. Lipoprotein lipase mRNA levels do not change in brown fat throughout pregnancy and lactation, whereas enzyme activity is significantly lowered. After 5 h of acute cold or noradrenaline administration there is a 2-fold increase in lipoprotein lipase mRNA abundance, whereas lipoprotein lipase activity is stimulated to more than 6-fold the basal values. It is concluded that translational and/or posttranslational mechanisms are involved in the noradrenergic modulation of lipoprotein lipase activity in brown fat.

Hepatectomy-nephrectomy effects in the pregnant rat and fetus

Levels of circulating glucose, glycerol, and FFA concentrations were determined before and after hepatectomy-nephrectomy in 20 day pregnant rats and virgin controls. After evisceration, blood glucose levels decreased in a parallel way in both groups whereas in pregnant rats, the blood glycerol level increased less and plasma-FFA rose more than in controls. Maternal evisceration caused reduced blood glucose and enhanced glycerol levels in fetuses, whereas fetal plasma-FFA levels were unmodified. Results indicate that extrahepatic glucose utilization remained stable in the late pregnant rat. Fetal levels of circulating glycerol, but not of FFA, appeared directly dependent on maternal levels. It is proposed that under normal conditions, glycerol availability to the fetus is low, due to its preferential utilization by maternal gluconeogenic organs which reduced the amount available for possible placental transfer.