Serotonin- and Dopamine-Related Gene Expression in db/db Mice Islets and in MIN6 ß-Cells Treated with Palmitate and Oleate.

High circulating nonesterified fatty acids (NEFAs) concentration, often reported in diabetes, leads to impaired glucose-stimulated insulin secretion (GSIS) through not yet well-defined mechanisms. Serotonin and dopamine might contribute to NEFA-dependent ß-cell dysfunction, since extracellular signal of these monoamines decreases GSIS. Moreover, palmitate-treated ß-cells may enhance the expression of the serotonin receptor Htr2c, affecting insulin secretion. Additionally, the expression of monoamine-oxidase type B (Maob) seems to be lower in islets from humans and mice with diabetes compared to nondiabetic islets, which may lead to increased monoamine concentrations. We assessed the expression of serotonin- and dopamine-related genes in islets from db/db and wild-type (WT) mice. In addition, the effect of palmitate and oleate on the expression of such genes, 5HT content, and GSIS in MIN6 ß-cell was determined. Lower Maob expression was found in islets from db/db versus WT mice and in MIN6 ß-cells in response to palmitate and oleate treatment compared to vehicle. Reduced 5HT content and impaired GSIS in response to palmitate (-25%; p < 0.0001) and oleate (-43%; p < 0.0001) were detected in MIN6 ß-cells. In conclusion, known defects of GSIS in islets from db/db mice and MIN6 ß-cells treated with NEFAs are accompanied by reduced Maob expression and reduced 5HT content.

Development and assessment of the disposition index based on the oral glucose tolerance test in subjects with different glycaemic status.

Insulin secretion and insulin sensitivity indexes are related by hyperbolic functions, allowing the calculation of the disposition index (DI) as the product of the acute insulin response (AIR) and the insulin sensitivity index (Si) from intravenous glucose tolerance test (IVGTT). Our objective was to develop an oral-DI based on the oral glucose tolerance test (OGTT) and to assess its association with glucose tolerance status. This research is structured in three studies. Study 1: OGTT were performed in 833 non-diabetic Chilean women (18-60 years) without family history of diabetes mellitus. Study 2: an independent group of n = 57 non-diabetic (18-46 years) without family history of diabetes mellitus carried out an OGTT and an abbreviated IVGTT. Study 3: a sample of 1674 Chilean adults (18-60 years) with different glycaemic status performed an OGTT. An adequate statistical fit for a rectangular hyperbola was found between the area under the curve of insulin-to-glucose ratio (AUCI/G-R) and the Matsuda ISI-COMP index (study 1). The oral-DI derived as AUCI/G-R × ISI-COMP was previously termed insulin-secretion-sensitivity index-2 (ISSI-2). ISSI-2 significantly correlated with DI from IVGTT (rho = 0.34; p = 0.009) (study 2). ISSI-2 shows important differences across groups of subjects with different glycaemic status (study 3). We have confirmed that ISSI-2 replicates the mathematical properties of DI, showing significant correlations with DI from the abbreviated MM-IVGTT. These results indicate that ISSI-2 constitutes a surrogate measure of insulin secretion relative to insulin sensitivity and emphasizes the pivotal role of impaired insulin secretion in the development of glucose homeostasis dysregulation.

Fluoxetine impairs insulin secretion without modifying extracellular serotonin levels in MIN6 ß-cells.

INTRODUCTION: Pancreatic ß-cells synthetize and store Serotonin (5-Hydroxytriptamine, 5HT) which is co-released with insulin. It has been proposed that extracellular 5HT binds to specific cell surface receptors and modulate insulin secretion. On the other hand, Selective Serotonin Reuptake Inhibitor (SSRI) fluoxetine seems to reduce Glucose-Stimulated Insulin Secretion (GSIS). However, it is unknown whether this effect results from changes in extracellular 5HT concentration owed to the blockade of 5HT transporter (SERT) or from non-5HT dependent actions. The aims of this work were: 1) to quantify extracellular 5HT levels and GSIS in ß-cell lines, 2) to determine whether extracellular 5HT levels and GSIS are changed by fluoxetine or 5-Hydroxytryptophan (5HTP, the immediate 5HT biosynthetic precursor), and 3) to quantify the expression of Slc6a4 gene (encoding SERT) in ß-cell lines in relation to other genes involved in 5HT system. MATERIAL AND METHODS: ß-cell lines MIN6 and RINm5f were subjected to GSIS protocols, after treatment with fluoxetine, 5HTP or 5HT. Insulin and 5HT were quantified by ELISA and HPLC, respectively. Relative mRNA expression was quantified by RT-qPCR. RESULTS: MIN6 ß-cells secretes 5HT in response to glucose, showing a sharp increase in 5HT release when cells were preloaded with 5HTP. Treatment with 5HT or fluoxetine reduces GSIS. Fluoxetine fails to further increases 5HTP-induced elevation of secreted 5HT. MIN6 ß-cells express both isoforms of Tryptophan Hydroxylase (Tph1 and Tph2), and have high expression levels of L-Dopa decarboxylase (Ddc), both enzymes involved in 5HT biosynthetic pathway, but do not express the 5HT transporters Slc6a4 or Slc6a3 (the Dopamine-5HT transporter) genes. CONCLUSION: The inhibitory effect of fluoxetine on ß-cell glucose stimulated insulin secretion is not mediated by blockage of 5HT transporter through SERT.

Plasma levels of interleukin-6 and interleukin-18 after an acute physical exercise: relation with post-exercise energy intake in twins.

Increased interleukin-6 (IL-6) plasma levels have been described to occur during physical exercise. A relative reduction in energy intake after physical activity has also been reported after exercise, indicating a possible involvement of IL-6 as an anorexigenic factor. Given the possible effect of interleukins on appetite, we assessed whether a controlled physical activity bout is related with changes in IL-6, IL-6 soluble receptor (IL-6sR), gp130 and interleukin-18 (IL-18) plasma levels, as well as their relation with post-exercise energy intake. A co-twin intervention study was carried out with five young male monozygotic twin pairs. One co-twin performed 45 min of submaximal exercise on a treadmill near the anaerobic threshold ending with 7 min at 90 % VO(2) max, while his co-twin remained non-active. Ad libitum energy intake was tested through a carbohydrate-rich meal test. Venous blood samples were drawn at baseline, immediately after exercise and after the meal ingestion. Plasma concentrations of IL-6, IL-6sR, gp130 and IL-18 were measured via ELISA. IL-6 plasma levels increased after physical activity bout (2.6-fold change; p = 0.04). A less marked trend, although still significant, was observed for plasma levels of IL-6sR and gp130. Plasma levels of IL-18 did not significantly change during exercise. The twins who exercised exhibited significantly lower energy intake (181 versus 1,195 kcal; p = 0.04), compared to the co-twins who remained resting. The present study in monozygotic twins shows increased IL-6 plasma levels after acute physical exercise with a significant reduction in energy intake, supporting a linkage between IL-6 and acute post-exercise eating behaviour.

Copy number polymorphism of the salivary amylase gene: implications in human nutrition research.

The salivary α-amylase is a calcium-binding enzyme that initiates starch digestion in the oral cavity. The α-amylase genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B) and a related pseudogene. The AMY1 genes show extensive copy number variation which is directly proportional to the salivary α-amylase content in saliva. The α-amylase amount in saliva is also influenced by other factors, such as hydration status, psychosocial stress level, and short-term dietary habits. It has been shown that the average copy number of AMY1 gene is higher in populations that evolved under high-starch diets versus low-starch diets, reflecting an intense positive selection imposed by diet on amylase copy number during evolution. In this context, a number of different aspects can be considered in evaluating the possible impact of copy number variation of the AMY1 gene on nutrition research, such as issues related to human diet gene evolution, action on starch digestion, effect on glycemic response after starch consumption, modulation of the action of α-amylases inhibitors, effect on taste perception and satiety, influence on psychosocial stress and relation to oral health.

Regulation of the A3 adenosine receptor gene in vascular smooth muscle cells: role of a cAMP and GATA element.

In previous studies, we reported that the level of expression of the adenylyl cyclase inhibitory A3 adenosine receptor (AR) impacts vascular tone and that rat vascular smooth muscle cells (VSMCs) coexpress the A3 AR and the adenylyl cyclase stimulatory A2a- and A2b-type ARs. In the current study, we investigated the regulation of expression of the A3 AR gene, focusing on sequences conserved in the mouse and human promoters. Transient transfection of primary cultures of rat VSMCs, using the mouse A3 AR promoter, shows that mutation of a conserved cAMP response element (CRE) significantly up-regulates promoter activity in first passage cells, whereas mutation of a conserved GATA site reduces promoter activity. This suggests that an inhibitory protein binds the CRE, whereas an enhancing factor binds the GATA sequence. Electrophoretic mobility shift assays (EMSAs) indicate that the putative CRE and GATA sites indeed bind cAMP response element modulator 1/c-Jun and the GATA6 protein, respectively. A3 AR promoter activity is significantly up-regulated in the presence of forskolin, the nonselective agonist 5'-(N-ethylcarboxamido)adenosine, or the A2a AR agonist 4-[2-[[6-amino-9(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepro- panoic acid (CGS21680), reaching levels similar to those of the A3 AR promoter bearing a mutated CRE. EMSA indicates that in the presence of forskolin the binding to the CRE is inhibited, suggesting that cAMP elevation disturbs the formation of an inhibitory complex on the CRE. Finally, semiquantitative reverse transcription-polymerase chain reaction analysis reveals that endogenous A3 AR mRNA is elevated in response to forskolin. Our findings suggest the presence of a mechanism by which cAMP might control its own level in cells via regulation of genes involved in modulation of adenylyl cyclase activity.

Formation of a phosphorus-phosphorus bond by successive one-electron reductions of a two-phosphinines-containing macrocycle: crystal structures, EPR, and DFT investigations.

Chemical and electrochemical reductions of the macrocycle 1 lead to the formation of a radical monoanion anion [1](*)(-) whose structure has been studied by EPR in liquid and frozen solutions. In accord with experimental (31)P hyperfine tensors, DFT calculations indicate that, in this species, the unpaired electron is mainly localized in a bonding sigma P-P orbital. Clearly, a one-electron bond (2.763 A) was formed between two phosphorus atoms which, in the neutral molecule, were 3.256 A apart (crystal structure). A subsequent reduction of this radical anion gives rise to the dianion [1](2)(-) which could be crystallized by using, in the presence of cryptand, Na naphthalenide as a reductant agent. As shown by the crystal structure, in [1](2)(-), the two phosphinine moieties adopt a phosphacyclohexadienyl structure and are linked by a P-P bond whose length (2.305(2) A) is only slightly longer than a usual P-P bond. When the phosphinine moieties are not incorporated in a macrocycle, no formation of any one-electron P-P bond is observed: thus, one-electron reduction of 3 with Na naphthalenide leads to the EPR spectrum of the ion pair [3](*)(-) Na(+); however, at high concentration, these ion pairs dimerize, and, as shown by the crystal structure of [(3)(2)](2)(-)[(Na(THF)(2))(2)](2+) a P-P bond is formed (2.286(2) A) between two phosphinine rings which adopt a boat-type conformation, the whole edifice being stabilized by two carbon-sodium-phosphorus bridges.

Inhibition of oncogene STAT3 phosphorylation by a prolactin antagonist, hPRL-G129R, in T-47D human breast cancer cells.

We have previously demonstrated that a hPRL antagonist (hPRL-G129R) was able to inhibit PRL induced breast cancer cell proliferation through induction of apoptosis. In the present study, we test the hypothesis that the inhibitory effect of hPRL-G129R in breast cancer cells occurs, at least in part, through the inhibition of oncogene STAT3 activation. We first demonstrated that STAT5 and STAT3 could be activated by either hGH or hPRL in T-47D breast cancer cells. Although the patterns of STAT5 activation by hGH and hPRL are similar, we observed a nearly 10-fold greater efficacy of hPRL in STAT3 activation as compared to that of hGH. More importantly, we have demonstrated that activation of STAT3 by hPRL could be inhibited by hPRL-G129R. Since T-47D cells coexpress GHR and PRLR, an attempt was made to dissect the molecular events mediated through hGHR or hPRLR using mouse L-cells expressing a single population of receptors (hGHR or hPRLR). To our surprise, only STAT5, not STAT3 phosphorylation was observed in these L-cells. In conclusion, our results suggest that: a) STAT3 is preferably activated through hPRLR in T-47D cells; b) hPRL-G129R is effective in inhibiting STAT3 phosphorylation; and c) the mechanism of STAT3 activation is different from that of STAT5.

Gold(I) and Gold(0) Complexes of Phosphinine-Based Macrocycles.

A "CO-like matrix", showing coordination analogous to that of carbonyl groups, is provided by silacalix[4]phosphinine macrocycles. Reaction with Au(I) leads to the first gold(I) complexes of macrocycles, which can be reduced with sodium or potassium to the paramagnetic gold(0) complexes (an example is shown), as evidenced by cyclic voltammetry and EPR spectroscopy.

A quantitative sucrose gradient analysis of the translational activity of 18 mRNA species in testes from adult mice.

Sucrose gradients have been widely used to study the translational activity of mRNA species in meiotic and haploid spermatogenic cells in mammals. Unfortunately, the results of these studies have been very inconsistent. The purpose of the present study was to obtain accurate and reproducible measurements of the translational activity of a large number of testicular mRNA in sucrose gradients. Extracts of adult testes and cultured seminiferous tubules were sedimented on sucrose gradients, and the distribution of 18 mRNA species was quantified by phosphoimaging. The proportions of various mRNA species sedimenting with polysomes in meiotic and haploid cells (approximately 6-74%) is less than typical of efficiently translated mRNAs (85-90%), demonstrating that the initiation of translation of virtually all mRNA species is at least partially inhibited and that the extent of inhibition is mRNA-specific. Most mRNA species in meiotic and early haploid spermatogenic cells are translated on polysomes in which the ribosome spacing is somewhat wider than in somatic cells, 100-150 verses 80-100 bases. However, the ribosome spacing on protamine mRNAs is unusually close (40-50 bases), and the spacing on poly(A) binding protein mRNA is unusually wide (212-272 bases), thus suggesting that the rate of translational initiation, termination and/or elongation is regulated on translationally active forms of certain mRNA.

Rat NAP1: cDNA cloning and upregulation by Mpl ligand.

The Mpl ligand is a hematopoietic cytokine which exerts its effects through association with the c-Mpl receptor. It regulates the proliferation, polyploidization and maturation of platelet precursors, the megakaryocytes. Using a differential display polymerase chain reaction (PCR) approach, we have identified an mRNA, belonging to a family of nucleosome assembly proteins, whose expression is upregulated in response to Mpl ligand. Multiple size classes of this mRNA (1.7, 2.5 and 4.3kb) are readily detected in rat primary bone marrow cells and hematopoietic tissues. The size classes are also expressed to different extents in cell lines of all hematopoietic lineages. We isolated the full-length cDNA encoding the rat megakaryocyte 1.7kb mRNA, referred to as rNAP1. Bacterially expressed recombinant protein encoded by the 1.7kb cDNA facilitates the formation of nucleosomes on relaxed circular DNA in vitro. Our data indicate that rNAPs, which may facilitate chromatin reorganization, are upregulated by Mpl ligand. It is possible that NAPs contribute to Mpl ligand's induced effects on hematopoietic cells.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Differential effects of heat shock on translation of normal mRNAs in primary spermatocytes, elongated spermatids, and Sertoli cells in seminiferous tubule culture.

Male germ cells in mice develop normally at 32 degrees C and spermatogenesis is severely inhibited by higher temperatures, including abdominal temperature, 37 degrees C. To examine the effects of heat stress on protein synthesis in various testicular cell types, seminiferous tubules were cultured at 32 degrees or 37 degrees C for 70 min or 42.5 degrees or 44 degrees C for 10 min followed by incubation for 60 min at 32 degrees C. Cultures were labeled with [35S]methionine, and the proteins that are soluble in 4% trichloroacetic acid were analyzed by acid-urea polyacrylamide gel electrophoresis. This culture system preserves the cytoarchitecture of the seminiferous epithelium and avoids breaking late haploid cells (elongated spermatids) during tissue dissociation. Incorporation of [35S]methionine into histone H1t, the testis-specific subtype of histone H1, in pachytene primary spermatocytes (meiotic cells) was reduced by about 33-50% following incubation at 37 degrees and 42.5 degrees C and by >/=90% after incubation at 44 degrees C. In contrast, exposure to 37 degrees, 42.5 degrees, and 44 degrees C had minimal effects on incorporation into transition proteins 1 and 2 in elongated spermatids. To determine whether heat stress inhibits translational initiation, the distribution of several mRNAs in cytoplasmic extracts of cultured tubules was analyzed by sucrose gradients and Northern blots. Exposure to 37 degrees and 44 degrees C produces incremental reductions in the size of polysomes translating H1t mRNA in pachytene spermatocytes and the sulfated glycoprotein 2 mRNA in Sertoli cells, the somatic cell type in the germinal epithelium. Neither 37 degrees nor 44 degrees C reduces the size or proportion of polysomal protamine 2 mRNA in elongated spermatids. These results demonstrate that the initiation of translation in pachytene spermatocytes and Sertoli cells is inhibited by exposure to abdominal temperature and that elongated spermatids are much more resistant to thermal stress.

Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

Use of P-wave-triggered, P-wave signal-averaged electrocardiogram to predict atrial fibrillation after coronary artery bypass surgery.

Atrial fibrillation occurs commonly after coronary artery bypass surgery. However, despite numerous attempts at prediction, no accurate and generally accepted method exists to predict its occurrence. P-wave-triggered P-wave signal averaging was performed on 54 patients before coronary artery bypass surgery to evaluate the utility of this method to predict atrial fibrillation after coronary artery bypass surgery. After excluding six patients with unevaluable P-wave signal averages and three patients with postoperative arrhythmias other than atrial fibrillation, the P-wave signal averages of 45 patients were analyzed. Sixteen patients had postoperative atrial fibrillation and 29 did not. The mean P-wave duration of the filtered, signal-averaged P wave was 163 +/- 19 msec in the 16 patients with atrial fibrillation and 144 +/- 16 msec in the 29 patients without (p < 0.005). Left atrial enlargement on the surface electrocardiogram (ECG) was the only other statistically significant variable that correlated weakly with the onset of postoperative atrial fibrillation (p = 0.04). Other clinical variables such as P-wave duration in ECG lead II, left ventricular hypertrophy on ECG, age, sex, hypertension, and left ventricular ejection fraction were not significantly different between the two groups. With a cut point of 155 msec, chi-squared analysis revealed a p value of < 0.005, yielding a sensitivity of 69%, a specificity of 79%, a positive predictive value of 65%, and a negative predictive value of 82%. Signal-averaging of the P wave in patients before coronary artery bypass surgery provides a good predictor of postoperative atrial fibrillation.