Cecilia Rojas

Essential fatty acids in early life: structural and functional role.

Essential fatty acids (EFA) are structural components of all tissues and are indispensable for cell membrane synthesis; the brain, retina and other neural tissues are particularly rich in long-chain polyunsaturated fatty acids (LCPUFA). These fatty acids serve as specific precursors for eicosanoids that regulate numerous cell and organ functions. Results from animal and recent human studies support the essential nature of n-3 EFA in addition to the well-established role of n-6 EFA for human subjects, particularly in early life. The most significant effects relate to neural development and maturation of sensory systems. Recent studies using stable-isotope-labelled tracers demonstrate that even preterm infants are able to form arachidonic acid (AA) and docosahexaenoic acid (DHA), but that synthesis is extremely low. Intracellular fatty acids or their metabolites regulate transcriptional activation of gene expression during adipocyte differentiation, and retinal and nervous system development. Regulation of gene expression by LCPUFA occurs at the transcriptional level and is mediated by nuclear transcription factors activated by fatty acids. These nuclear receptors are part of the steroid hormone receptor family. Two types of polyunsaturated fatty acid responsive transcription factors have been characterized, the peroxisome proliferator-activated receptor (PPAR) and the hepatic nuclear factor 4alpha. DHA also has significant effects on photoreceptor membranes involved in the signal transduction process, rhodopsin activation, and rod and cone development. Comprehensive clinical studies have shown that dietary supplementation with marine oil or single-cell oils, sources of LCPUFA, results in increased blood levels of DHA and AA, as well as an associated improvement in visual function in formula-fed premature infants to match that of human milk-fed infant. Recent clinical trials convincingly support LCPUFA supplementation of preterm infant formulations and possibly term formula to mimic human milk composition.

Human cord blood-derived mesenchymal stem cells home and survive in the marrow of immunodeficient mice after systemic infusion.

Bone marrow is the residence site of mesenchymal stem cells (MSC), which upon commitment and maturation develop into several mesenchymal phenotypes. Recently, we have described the presence of MSC in human cord blood (cbMSC) and informed that their properties are the same as those for MSC obtained from adult bone marrow. In this study we have investigated the capability of transplanted cbMSC to home and survive in the marrow of unconditioned nude mice. cbMSC utilized for transplantation studies were characterized by morphology, differentiation potential, and immunophenotype. After transplantation by systemic infusion, human DNA (as detected by PCR amplification of human-specific β-globin gene) was detected in the marrow of recipients as well as in ex vivo-expanded stromal cells prepared from the marrow of transplanted animals. These results demonstrate homing and survival of cbMSC into the recipient marrow and also suggest a mesenchymal-orientated fate of engrafted cells, because human DNA was also detected in cells of other recipient tissues, like cardiac muscle, teeth, and spleen.

Biosynthesis of monoterpene hydrocarbons from [1-3H]neryl pyrophosphate and [1-3H]geranyl pyrophosphate by soluble enzymes from Citrus limonum

Abstract A soluble enzyme preparation from the flavedo of Citrus limonum transforms [1- 3 H 1 ]neryl pyrophosphate or [1- 3 H 1 ]geranyl pyrophosphate into β-pinene, sabinene, α-pinene, and limonene. The enzyme has been partially purified and stabilized by precipitation with polyethyleneglycol. The enzymic cyclization requires the presence of Mn 2+ , which cannot be replaced with Mg 2+ . The addition of reagents containing sulfhydryl groups is essential for optimal activity. Allylic C 10 monophosphates do not act as substrates, but they inhibit hydrocarbon formation. Inorganic pyrophosphate has a similar inhibitory effect. No interconversion of neryl and geranyl pyrophosphate has been observed. Possible pathways for the enzymic cyclization reactions are proposed.

The anti-adipogenic effect of angiotensin II on human preadipose cells involves ERK1,2 activation and PPARG phosphorylation

Despite the importance of adipocyte formation for adipose tissue physiology, current knowledge about the mechanisms that regulate the recruitment of progenitor cells to undergo adipogenic differentiation is limited. A role for locally generated angiotensin II emerged from studies with human and murine cells. Preadipose cells from different human fat depots show reduced response to adipogenic stimuli when exposed to angiotensin II. This investigation sought to gain an insight into the intracellular mechanisms involved in the anti-adipogenic response of human preadipose cells from omental fat to angiotensin II. Its effect was evaluated on cells stimulated to adipogenic differentiation in vitro, by assessment of glycerol-3-phosphate dehydrogenase activity and expression of early markers of adipogenesis. Extracellular signal-regulated kinase(1,2) (ERK(1,2)) pathway activation was inferred from the phosphorylated to total ERK(1,2) ratio determined by western blot. Exposure to angiotensin II throughout the 10-day differentiation period resulted in a reduced adipogenic response. A similar anti-adipogenic effect was observed when this hormone was present during the first 48 h of induction to differentiation. Angiotensin II treatment had no consequences on CCAAT/enhancer-binding protein beta and peroxisome proliferator-activated receptor gamma (PPARG) induction, but increased the phosphorylated form of the key adipogenic regulator PPARG. Upon angiotensin II exposure, a raise of phosphorylated ERK(1,2) was determined, which was more prominent 8-20 h after induction of adipogenesis (when controls reached negligible values). Chemical inhibition of ERK(1,2) phosphorylation prevented angiotensin II-dependent reduction in adipogenesis. These results support the participation of the mitogen-activated protein kinase/ERK(1,2) pathway in the anti-adipogenic effect of angiotensin II on preadipose cells from human omental adipose tissue.

Antilipolytic effect of calcium-sensing receptor in human adipocytes

The extracellular calcium-sensing receptor (CaSR), a seven transmembrane G-protein-coupled receptor, was cloned in 1993. Its activation was first associated to the regulation of calcium homeostasis; however, the presence in tissues unrelated with this role has revealed its participation in numerous other cell functions. We previously described CaSR expression in human adipocytes, and here we investigated the effect of its activation on adipocyte lipolytic activity by measuring glycerol release to the incubation medium. Treatment of adipocytes with CaSR agonists elicited an inhibitory effect on basal lipolysis, which was prevented by a CaSR antagonist. To further corroborate the antilipolytic effect of CaSR activation, lipolysis was evaluated under conditions that interfere with main antilipolytic regulatory pathways. Cells were preincubated with pertussis toxin (PT, a Giα protein inhibitor), the phosphatidylinositol 3 kinase (PI3K) inhibitors wortmannin and LY-294002 as well as the cAMP analog 8Br-cAMP, all of which influenced the antilipolytic effect of CaSR stimulation. In light of the current view of adipose tissue as an organ involved in whole-body metabolic control, the role of the CaSR modulating basal lipolysis elicits great interest, given its metabolic sensing capabilities due to the variety of ligands that regulate its activity, and its potential cross-talk with insulin and adipose tissue-secreted factors.

Larger Anti-adipogenic Effect of Angiotensin II on Omental Preadipose Cells of Obese Humans

Objective: The ability to form new adipose cells is important to adipose tissue physiology; however, the mechanisms controlling the recruitment of adipocyte progenitors are poorly understood. A role for locally generated angiotensin II in this process is currently proposed. Given that visceral adipose tissue reportedly expresses higher levels of angiotensinogen compared with other depots and the strong association of augmented visceral fat mass with the adverse consequences of obesity, we studied the role of angiotensin II in regulating adipogenic differentiation in omental fat of obese and non‐obese humans.

Enhancement of the hydrolysis of geranyl pyrophosphate by bivalent metal ions. A model for enzymic biosynthesis of cyclic monoterpenes

Hydrolysis of geranyl pyrophosphate is catalyzed by salts of Mn2+ and involves C-O bond cleavage. The first order rate constants reach limiting values with [Mn2+] > 10−2 M, and the most reactive species is GPP(Mn2+)2 at the optimum pH of 6.5–7. The products are similar to those from acid hydrolysis except that more cyclic hydrocarbons are formed in the presence of metal ions. Hydrolysis of geranyl phosphate is inhibited, and that of citronnellyl pyrophosphate is weakly catalyzed by Mn2+. Other divalent metal cations catalyze the hydrolysis of geranyl pyrophosphate and the sequence of effectiveness is Cu2+ > Mn2+ > Zn2+ > Co2+ < Mg2+ ~ Ca2+.

Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line ☆

Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.

The effect of overweight and obesity on proliferation and activation of AKT and ERK in human endometria.

OBJECTIVE To examine whether overweight and obesity could lead to increased endometrial proliferation and activation of AKT and ERK1,2 in cycling premenopausal women. METHODS Endometrial and blood samples were obtained from women with normal endometrial histology, and allocated into three groups-normal-weight, overweight and obese-according to the subjects body mass index (BMI). Samples from obese patients with type-I endometrial cancer (EC) were included as a control. Cell proliferation was measured by immunohistochemical detection of Ki67 and phosphorylated histone H3 (p-H3). AKT and ERK1,2 activation was assessed by Western blot. Circulating steroids, leptin and insulin were measured by immunoassays. RESULTS In endometrial samples with normal histology, epithelial cell proliferation was higher in the overweight and obese groups versus the normal-weight set (P<0.05). Proliferation indexes were positively correlated with the subjects BMI and serum levels of estrogen, leptin and insulin (P<0.05). Increased phosphorylated AKT (pAKT) (1.6-fold) and ERK1,2 (pERK1,2) (8.7-fold) were observed in endometria from obese with respect to normal-weight subjects (P<0.05). Similarly, increased phosphorylation of AKT (0.7-fold) and ERK1,2 (2.3-fold) was detected in endometria from overweight as compared with the normal-weight group (P<0.05). In women with EC, we found a significant increase in endometrial proliferation, and in pAKT and pERK1,2 expression levels when compared to patients with normal endometrial histology. CONCLUSION These results show correlation between obesity (and overweight) and increased endometrial cell proliferation, and the activation of AKT and ERK1,2. These features could be related with the higher risk to develop type-I EC in overweight and obese women.

Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel

Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant alpha-subunits with the beta1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant α-subunits with the β1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.