Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotor activity.

White adipose tissue is known to contain the components of the renin-angiotensin system, which gives rise to angiotensin II from angiotensinogen (AGT). Recent evidence obtained in vitro and ex vivo is in favor of angiotensin II acting as a trophic factor of adipose tissue development. To determine whether AGT plays a role in vivo in this process, comparative studies were performed in AGT-deficient (agt(-/-)) mice and control wild-type mice. The results showed that agt(-/-) mice gain less weight than wild-type mice in response to a chow or high fat diet. Adipose tissue mass from weaning to adulthood appeared altered rather specifically, as both the size and the weight of other organs were almost unchanged. Food intake was similar for both genotypes, suggesting a decreased metabolic efficiency in agt(-/-) mice. Consistent with this hypothesis, cellularity measurement indicated hypotrophy of adipocytes in agt(-/-) mice with a parallel decrease in the fatty acid synthase activity. Moreover, AGT-deficient mice exhibited a significantly increased locomotor activity, whereas metabolic rate and mRNA levels of uncoupling proteins remained similar in both genotypes. Thus, AGT appears to be involved in the regulation of fat mass through a combination of decreased lipogenesis and increased locomotor activity that may be centrally mediated.

Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of CCAAT/enhancer binding proteins beta and -delta in preadipocytes.

The essential role of CCAAT/enhancer binding proteins (C/EBPs) beta and delta for adipocyte differentiation has been clearly established. In preadipocytes, their expression is up-regulated by the activation of leukemia inhibitory factor receptor (LIF-R) and prostacyclin receptor (IP-R) via the extracellular signal-regulated kinase (ERK) pathway and cAMP production, respectively. However, the molecular mechanisms by which LIF and prostacyclin-induced signals are propagated to the nucleus and the transcription factors mediating ERK and cAMP-induced C/EBP gene expression were unknown. Here we report that both pathways share cAMP responsive element binding protein/activation transcription factor 1 (CREB/ATF-1) as common downstream effectors. LIF-R and IP-R activation induced binding of CREB and/or ATF-1 to C/EBP promoters and CREB-dependent transcription. Expression of dominant negative forms of CREB dramatically reduced the LIF- and prostacyclin-stimulated C/EBP beta and C/EBP delta expression. Upon stimulation of the IP-R, the ERK pathway was activated in a PKA-dependent manner. ERK activation by the PKA pathway was not required for CREB/ATF-1 phosphorylation but rather was necessary for CREB-dependent up-regulation of C/EBPs expression. Our findings suggest that ERK activation is required for CREB transcriptional activity, possibly by recruitment of a coactivator.

Angiotensin II as a trophic factor of white adipose tissue: stimulation of adipose cell formation.

White adipose tissue is known to contain the components of the renin-angiotensin system giving rise to angiotensin II (AngII). In vitro, prostacyclin is synthesized from arachidonic acid through the activity of cyclooxygenases 1 and 2 and is released from AngII-stimulated adipocytes. Prostacyclin, in turn, is able to favor adipocyte formation. Based upon in vivo and ex vivo experiments combined to immunocytochemical staining of glycerol-3-phosphate dehydrogenase (GPDH), an indicator of adipocyte formation, it is reported herein that AngII favors the appearance of GPDH-positive cells. In the presence of a cyclooxygenase inhibitor, this adipogenic effect is abolished, whereas that of (carba)prostacyclin, a stable analog of prostacyclin that bypasses this inhibition, appears unaltered. Taken together, these results are in favor of AngII acting as a trophic factor implicated locally in adipose tissue development. It is proposed that AngII enhances the formation of GPDH-expressing cells from preadipocytes in response to prostacyclin released from adipocytes.

Angiotensinogen, angiotensin II and adipose tissue development.

Adipose tissue is an important source of angiotensinogen (AGT). Recent evidence shows that a local renin-angiotensinogen system (RAS) is present in human adipose tissue and may act as a distinct system from plasma RAS. In obese patients, the involvement of angiotensin II (angII) as a consequence of increased plasma AGT secreted from adipose tissue has been proposed in the development of hypertension. Another role of AGT via angII in the development of adipose tissue is supported by the following: (i) in vitro, angII stimulates the production and release of prostacyclin from adipocytes, which in turn promotes the differentiation of precursor cells into adipocytes; (ii) ex vivo and in vivo, both angII and (carba)prostacyclin promote the formation of new fat cells; and (iii) AGT -/- mice exhibit a slowing down of adipose tissue development, as compared to wild-type mice. Altogether the data are consistent with an autocrine/paracrine mechanism implicating AGT, angII and prostacyclin in adipose tissue development.

Prostacyclin IP receptor up-regulates the early expression of C/EBPbeta and C/EBPdelta in preadipose cells.

Prostacyclin (PGI(2)) and its stable analogue carbacyclin (cPGI(2)) are known to trigger the protein kinase A pathway after binding to the cell surface IP receptor and to promote or enhance terminal differentiation of adipose precursor cells to adipose cells. The early expression of C/EBPbeta and C/EBPdelta is known to be critical for adipocyte differentiation in vitro as well as in vivo. We report herein that in Ob1771 and 3T3-F442A preadipose cells, activation of the IP receptor by specific agonists (PGI(2), cPGI(2) and BMY 45778) is sufficient to up-regulate rapidly the expression of C/EBPbeta and C/EBPdelta. Cyclic AMP-elevating agents are able to substitute for IP receptor agonists, in agreement with the coupling of IP receptor to adenylate cyclase. Consistent with the fact that PGI(2) is released from preadipose cells and behaves as a paracrine/autocrine effector of adipose cell differentiation, the present results favor a key role of prostacyclin by means of the IP receptor and its intracellular signaling pathway in eliciting the critical early expression of both transcription factors.

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). In preadipose cells, carbacyclin and alpha-bromopalmitate are active and BRL49653 shows no effect, whereas all these ligands are active in adipose cells. The stimulatory effect of PPAR agonists is potentiated by RXR agonists in adipose cells. In contrast to the UCP-1 gene, norepinephrine as a cAMP-elevating agent does not enhance the expression of UCP-2 gene. Altogether, the data favor a predominant role of PPARdelta in preadipose cells and the involvement of PPARgamma2 in adipose cells in up-regulating UCP-2 gene expression. Thus, a potential link between fatty acid metabolism and thermogenesis may exist in PPAR-expressing tissues.

Modulation of vascular tone and glycerol levels measured by in situ microdialysis in rat adipose tissue.

Changes in blood flow induced by the beta-adrenoceptor agonist isoproterenol (Iso) and a stable analogue of the major metabolite of arachidonic acid in adipose tissue, carbaprostacyclin (cPGI2), have been studied in rat periepididymal fat pad with in situ microdialysis measuring the distribution ratio of 0.2% ethanol in the dialysate (outflow) to that in the perfusate (inflow) (O/I ratio). Local perfusions of 1 microM cPGI2 or 1 microM Iso led to reversible decreases of the O/I ratio that were similar to the decrease induced by the vasodilating reference drug hydralazine (Hydra, 630 microM). Interestingly, a continuous perfusion of Hydra at a submaximal vasodilating concentration (63 microM) was sufficient to prevent further vasodilatation induced by Iso or cPGI2. To take advantage of this observation, experiments were designed to evaluate the influence of the vasodilating effect of Iso or cPGI2 on the ability of either to induce lipolysis in vivo. The results showed that the vasodilating effect of Iso could contribute to glycerol removal from the extracellular fluid and demonstrate that cPGI2 was devoid of lipolytic activity.

Effects of hyposmotic shock on taurine transport in isolated trout hepatocytes.

Isolated trout hepatocytes exposed to hypotonic medium undergo a regulatory volume decrease (RVD) that occurs via two separate routes, K(+)-Cl- cotransport and amino acid release, the ion efflux accounting for 70% of the total osmolyte loss. Taurine, glutamine and glutamic acid are the most important and represent 73% of the total amino acid content (53 mmol (l cell water)-1). The osmolarity-sensitive release of amino acids was studied using [3H]taurine. Kinetic studies indicated two components for taurine influx: a linear Na(+)-independent transport and a saturable Na(+)-dependent system with a Michaelis-Menten constant (Km) of 122 microM and a maximum velocity (Vmax) of 31.2 pmol (mg protein)-1 min-1. This second way of uptake was also chloride dependent and indicated an apparent coupling ratio Na+:Cl-:taurine of 2:1:1. The latter component and the taurine efflux were stimulated during RVD, leading to intracellular amino acid loss. Taurine efflux activation during volume recovery was transient and also dependent on the presence of both Na+ and Cl- in the extracellular medium. Furthermore, taurine release and RVD were slowed down when Ca2+ was omitted from the medium. These results suggested two distinct and complementary mechanisms for volume regulation in trout hepatocytes during hypotonic conditions.