GDM-associated insulin deficiency hinders the dissociation of SERT from ERp44 and down-regulates placental 5-HT uptake.

Serotonin (5-HT) transporter (SERT) regulates the level of 5-HT in placenta. Initially, we found that in gestational diabetes mellitus (GDM), whereas free plasma 5-HT levels were elevated, the 5-HT uptake rates of trophoblast were significantly down-regulated, due to impairment in the translocation of SERT molecules to the cell surface. We sought to determine the factors mediating the down-regulation of SERT in GDM trophoblast. We previously reported that an endoplasmic reticulum chaperone, ERp44, binds to Cys200 and Cys209 residues of SERT to build a disulfide bond. Following this posttranslational modification, before trafficking to the plasma membrane, SERT must be dissociated from ERp44; and this process is facilitated by insulin signaling and reversed by the insulin receptor blocker AGL2263. However, the GDM-associated defect in insulin signaling hampers the dissociation of ERp44 from SERT. Furthermore, whereas ERp44 constitutively occupies Cys200/Cys209 residues, one of the SERT glycosylation sites, Asp208 located between the two Cys residues, cannot undergo proper glycosylation, which plays an important role in the uptake efficiency of SERT. Herein, we show that the decrease in 5-HT uptake rates of GDM trophoblast is the consequence of defective insulin signaling, which entraps SERT with ERp44 and impairs its glycosylation. In this regard, restoring the normal expression of SERT on the trophoblast surface may represent a novel approach to alleviating some GDM-associated complications.

Down-regulation of the serotonin transporter in hyperreactive platelets counteracts the pro-thrombotic effect of serotonin.

An elevated plasma concentration of serotonin ([5-HT]) is a common feature of cardiovascular disease often associated with enhanced platelet activation and thrombosis. Whether elevated in vivo plasma 5-HT per se represents an independent risk factor for platelet hyperreactivity or only is an epiphenomenon of cardiovascular disease is poorly understood. We examined in vitro and in vivo platelet function following a 24h elevation of plasma [5-HT] in mice. In vivo administration of 5-HT using osmotic minipumps increased plasma [5-HT] in treated mice compared to control mice instrumented with saline loaded pumps. 5-HT infusion did not increase systolic blood pressure, but markers of platelet activation including P-selectin and (PE)Jon/A staining were increased and these findings coincided with the enhanced aggregation of isolated platelets in response to type I fibrillar collagen. Tail bleeding times and the time to occlusion following chemical damage to the carotid artery were shortened in 5-HT-infused mice. 5-HT-infused mice were treated with paroxetine (Prx) to block 5-HT uptake via the serotonin transporter (SERT). Prx lowered platelet [5-HT] and attenuated platelet activation and aggregation. These results and our biochemical indices of enhanced 5-HT intracellular signaling in the platelets of 5-HT-infused mice reveal a mechanistic link between elevated plasma [5-HT], abnormal intracellular 5-HT signaling and accentuated platelet aggregation. Although a down-regulation of the serotonin transporter (SERT) on the platelet surface may counteract the pro-thrombotic influence of elevated plasma [5HT], this compensatory mechanism may fail to prevent the increased thrombotic risk caused by elevated plasma [5-HT].

Evaluation of task shifting in community-based DOTS program as an effective control strategy for tuberculosis.

Tuberculosis (TB) remains to be the most prevalent and debilitating pulmonary (PTB) infection in the world today, affecting about one-third of the world's population. TB is an endemic disease in many developing countries, and efforts at eliminating the disease remain futile. While the course of the disease is indolent with years of latency, the reactivation of the disease can pose serious pulmonary and systemic infections that compromise multiple organ functions which lead to respiratory failure or end-organ damage. Despite attempts to control and eradicate the mycobacterium, the prevalence of the disease remains high due to increasing population rate, persistence of poverty and poor health care, treatment failure, increasing multidrug resistance as a consequence of treatment failure and poor compliance, and existence of comorbid conditions that compromise immune response. Limited government resources to screen and monitor disease progression of TB in third world countries hamper the eradication of the disease. In response, we have evaluated the efficiency and effectivity of a Community-Based Directly Observed Treatment, Short-Course (CB-DOTS), which is an equally effective alternative strategy to health center DOTS.

Effect of serotonin on platelet function in cocaine exposed blood.

5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine.

Enhanced gastrointestinal expression of cytosolic malic enzyme (ME1) induces intestinal and liver lipogenic gene expression and intestinal cell proliferation in mice.

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue's metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.

Impact of elevated plasma serotonin on global gene expression of murine megakaryocytes.

BACKGROUND: Serotonin (5-HT) is a biogenic amine that also acts as a mitogen and a developmental signal early in rodent embryogenesis. Genetic and pharmacological disruption of 5-HT signaling causes various diseases and disorders via mediating central nervous system, cardiovascular system, and serious abnormalities on a growing embryo. Today, neither the effective modulators on 5-HT signaling pathways nor the genes affected by 5-HT signal are well known yet. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to identify the genes altered by 5-HT signaling pathways, we analyzed the global gene expression via the Illumina array platform using the mouse WG-6 v2.0 Expression BeadChip containing 45,281 probe sets representing 30,854 genes in megakaryocytes isolated from mice infused with 5-HT or saline. We identified 723 differentially expressed genes of which 706 were induced and 17 were repressed by elevated plasma 5-HT. CONCLUSIONS/SIGNIFICANCE: Hierarchical gene clustering analysis was utilized to represent relations between groups and clusters. Using gene ontology mining tools and canonical pathway analyses, we identified multiple biological pathways that are regulated by 5-HT: (i) cytoskeletal remodeling, (ii) G-protein signaling, (iii) vesicular transport, and (iv) apoptosis and survival. Our data encompass the first extensive genome-wide based profiling in the progenitors of platelets in response to 5-HT elevation in vivo.

A serotonin-induced N-glycan switch regulates platelet aggregation.

Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.

Data retention after a patient withdraws consent in clinical trials.

Patient retention is critically important in the conduct of a successful clinical trial. The power in numbers in multicenter trials is dependent on the completion of follow-up for every patient randomized. If at the end of a clinical trial, a significant number of randomized patients are missing outcome data, there will not be enough pool for data analyses to conclude a study based on its primary and secondary objectives. When patients who are either lost to follow-up or who withdraw consent during the clinical trial are eliminated from the data pool, they subsequently affect the power and the validity of conclusions derived from the clinical study. This paper aims to present current guidance on data retention for patients who have withdrawn consent from clinical trials.

Communication between 5-HT and small GTPases.

Advances over the past decade have improved our understanding of the serotonin (5-HT) biology outside the central nervous system specifically the molecular mechanisms of serotonergic signaling in association with small GTPases. It is now recognized that the communication between 5-HT and GTPases plays important roles in peripheral tissues, vascular cells and are involved in coagulation, hypertension, inflammation, healing and protection. Furthermore, 5-HT receptors as heterotrimeric GTP-binding protein-coupled receptors act as effector protein on the small GTPases. Therefore, the antagonists or agonists of the effector proteins of small GTPases could be useful therapeutic agents for the treatment of several diseases and disorders.

Molecular mechanisms of SERT in platelets: regulation of plasma serotonin levels.

The serotonin transporter (SERT) on platelets is a primary mechanism for serotonin (5HT) uptake from the blood plasma. Alteration in plasma 5HT level is associated with a number of cardiovascular diseases and disorders. Therefore, the regulation of the transporter's activity represents a key mechanism to stabilize the concentration of plasma 5HT. There is a biphasic relationship between plasma 5HT elevation, loss of surface SERT, and depletion of platelet 5HT. Specifically, in platelets, plasma membrane SERT levels and platelet 5HT uptake initially rise as plasma 5HT levels are increased but then fall below normal as the plasma 5HT level continues to rise. Therefore, we propose that elevated plasma 5HT limits its own uptake in platelets by down-regulating SERT as well as modifying the characteristics of SERT partners in the membrane trafficking pathway. This review will summarize current findings regarding the biochemical mechanisms by which elevated 5HT downregulates the expression of SERT on the platelet membrane. Intriguing aspects of this regulation include the intracellular interplay of SERT with the small G protein Rab4 and the concerted 5HT-mediated phosphorylation of vimentin.

Soy protein diet alters expression of hepatic genes regulating fatty acid and thyroid hormone metabolism in the male rat.

We hypothesized that consumption of soy protein isolate (SPI) or the soy isoflavone genistein (GEN) would modulate mRNA expression of genes underlying lipid and thyroid hormone metabolism in livers and small intestines of young adult male Sprague-Dawley rats. Early pregnant rat dams were placed on AIN-93G diets containing casein (CAS, control protein), SPI, or CAS+GEN. Litters were weaned to the same diet as their dam. SPI-fed (but not GEN-fed) male rats of 48 days of age had significant reductions in body weight, abdominal fat pad weight and hepatic content of lipid droplets and triglycerides. Hepatic peroxisome proliferator-activated receptor α (Ppara) transcripts were elevated with SPI but not GEN diet. Hepatic pyruvate dehydrogenase kinase-4 (Pdk4) and cytochrome P450 4A10 (Cyp4a10) mRNA abundance was reduced with SPI; the SPI effect on Cyp4a10 was recapitulated by GEN diet. SPI (but not GEN) suppressed Pdk4 and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) mRNA abundance in duodenum. Liver iodothyronine deiodinase types 1 and 2 (Dio1 and Dio2) mRNA levels were increased with SPI diet; the effect on Dio2, but not Dio1 mRNAs, also was observed with GEN. SPI and GEN increased hepatic types 1 and 2 iodothyronine deiodinase (D1 and D2) activities. Effects of SPI and GEN on the above gene expression may contribute to the observed reductions in body and adipose tissue weight and liver lipid content in this model. Identification of the regulation, by genistein and soy protein, of iodothyronine deiodinase synthesis has potential applications for treatment and prevention of fatty liver disease and obesity.