Co-expression of NPY and VIP in human dental pulp

Introduction: The regulation of pulpal haemodynamics in health and disease involves sympathetic and parasympathetic mechanisms in which both neuropeptide Y (NPY; a sympathetic vasoconstrictor) and vasoactive intestinal polypeptide (VIP; a parasympathetic vasodilator) may play potential pathophysiological roles. We have previously investigated the levels of NPY or VIP present in human dental pulp tissue and shown that their expression is up-regulated in caries induced pulpal inflammation. Objectives: The aim of this study was to investigate the potential correlation between NPY and VIP levels measured in the same dental pulp samples using radioimmunoassay (RIA). Methods: Pulp tissue was obtained from extracted teeth, classified as follows; healthy (n=22), moderately carious (n=20) and grossly carious (n=26). Samples were processed for RIA by boiling in acetic acid as previously described. The levels of NPY and VIP, measured by RIA, were expressed as ng/gram of pulp tissue. The nature of the relationship between NPY and VIP levels in human pulp tissue was tested by calculating Pearson's product moment correlation coefficient using the linear regression test. Results: Calculation of Pearson product moment correlation coefficient showed a significant negative correlation between NPY and VIP levels in pulp tissue samples from non-carious teeth (p = 0.02, r = -.48). This negative correlation in non-carious teeth changed to a significant positive correlation in carious teeth when the levels of NPY and VIP were compared (p = 0.03, r= 0.311). Conclusions: In non-carious teeth, the negative correlation between NPY and VIP levels is in keeping with the previously described modulatory influence of cholinergic nerves on sympathetic function which may be perturbed as caries develops.

Multiple Markers of Inflammation in Crevicular Fluid During Experimental Gingivitis

Objective: To evaluate temporal changes in GCF levels of substance P, cathepsin G, interleukin 1 beta (IL-1&beta), neutrophil elastase and alpha1-antitrypsin (&alpha1AT) during development of and recovery from experimental gingivitis. Methods: Healthy human volunteers participated in a split-mouth study: experimental gingivitis was induced using a soft vinyl splint to cover test teeth during brushing over 21 days, after which normal brushing was resumed. Modified gingival index (MGI), gingival bleeding index (BI) and modified Quigley and Hein plaque index (PI) were assessed and 30-second GCF samples taken from 4 paired test and contra-lateral control sites in each subject at days 0, 7, 14, 21, 28 and 42. GCF volume was measured and site-specific quantification of one analyte per GCF sample was performed using radioimmunoassay (substance P), enzyme assay (cathepsin G) or ELISA (IL-1&beta, elastase, &alpha1AT). Site-specific data were analysed using analysis of repeated measurements and paired sample tests. Results: 56 subjects completed the study. All measurements at baseline (day 0) and at control sites throughout the study were low. Clinical indices and GCF volumes at the test sites increased from day 0, peaking at day 21 (difference between test and control for PI, BI, MGI and GCF all p<0.0001) and decreased again to control levels by day 28. Levels of four inflammatory markers showed a similar pattern, with significant differences between test and control apparent at 7 days (substance P p=0.0015; cathepsin G p=0.029; IL-1&beta p=0.026; elastase p=0.0129) and peaking at day 21 (substance P p=0.0023; cathepsin G, IL-1&beta and elastase all p<0.0001). Levels of &alpha1AT showed no apparent pattern over the course of the study. Conclusion: GCF levels of substance P, cathepsin G, IL-1&beta and neutrophil elastase have the potential to act as early markers of experimentally-induced gingival inflammation.

Vasoactive Intestinal Polypeptide (VIP) in Human Dental Pulps

Pulpal innervation is not exclusively sensory and there are potential roles for other neuropeptides such as vasoactive intestinal polypeptide (VIP) in pulpal health and disease. In the systemic circulation VIP relaxes vascular smooth muscles leading to vasodilatation. It has been shown that VIP fibres are associated with pulpal blood vessels and therefore VIP may mediate vasoactivity in the dental pulp. A growing body of evidence has now demonstrated that an additional major physiological role of VIP is to act as a survival factor. In order to gain a better understanding of the role of neuropeptides in the caries process it is of interest to specifically examine a role for VIP. Objectives: The aim of the present study was to determine the levels of VIP in carious (moderately carious and grossly carious) compared with non-carious teeth. Methods: A total of 68 teeth were included in the study (22 non-carious, 20 moderately carious and 26 grossly carious). VIP was measured in all samples using a sensitive and specific radioimmunoassay. Results: The mean concentration of VIP in the pulps of non-carious teeth was 7.69 ng/g (9.41 SD) compared to 14.93 ng/g (15.58 SD) in carious teeth. Pair-wise comparisons of VIP levels using Tukey’s test showed statistically significant differences in VIP expression between non-carious and moderately carious teeth (p=0.002) and between moderately and grossly carious teeth, (p=0.002). Conclusion: The significantly increased levels of VIP in moderately carious pulps compared with either non-carious or grossly carious pulps may suggest a role for VIP as a protective or survival factor.

Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein.

The production and puriWcation of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1–125)] using an Escherichia coli system and one step puriWcation process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1–125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1–125) synthesis was induced by addition of 1mM isopropyl- -D-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1–125) as judged by SDS–PAGE and yielded up to 40mg/L of culture medium (3.3mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1–125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1–34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera speciWc for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay speciWc for piscine N-terminal (1–34 aa) PTHrP, the recombinant sbPTHrP(1–125) was equipotent with PTHrP(1–34) in displacing labelled 125I-PTHrP(1–36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region speciWc assays and studies aimed at deWning post-secretory processing of this protein and its biological activity in Wsh.

Carcinoembryonic antigen (CEA) in non digestive cancerous and normal tissues.

Carcinoembryonic antigen (CEA), immunologically identical to CEA derived from colonic carcinoma, was identified and purified from perchloric acid (PCA) extracts of bronchial and mammary carcinoma. CEA extracted from bronchial and mammary carcinoma was quantitated by single radial immunodiffusion and was found to be in average about 50-75 times less abundant in these tumors than in colonic carcinoma. CEA could also be detected in one normal breast in lactation and at lower concentrations in normal lung (1000-4000 times lower than in colonic carcinoma). The small amounts of CEA present in normal tissues are distinct from the glycoprotein of small mol. wt showing only partial identity with CEA, that we recently identified and extracted in much larger quantities from normal lung and spleen. The demonstration of the presence of CEA in non digestive carcinoma by classical gel precipitation analysis suggests that the CEA detected in the plasma of such patients by radioimmunoassay is also identical to colonic carcinoma CEA. Our comparative study of plasma CEA from bronchial and colonic carcinoma, showing that CEA from both types of patient has the same elution pattern on Sephadex G-200 and gives parallel inhibition curves in the radioimmunoassay, is in favor of this hypothesis. However, it should not be concluded that all positive CEA radioimmunoassay indicate the presence of an antigen identical to colonic carcinoma CEA. A word of warning concerning the interpretation of radioimmunoassay is required by the observation that the addition of mg amounts of PCA extract of normal plasma, cleared of CEA by Sephadex filtration, could interfere in the test and mimic the presence of CEA.

Use of limitations of radiolabeled anti-CEA antibodies and their fragments for photoscanning detection of human colorectal carcinomas.

Fifty-three patients with histologically proven carcinoma were injected with highly purified [131I]-labeled goat antibodies or fragments of antibodies against carcinoembryonic antigen (CEA). Each patient was tested by external photoscanning 4, 24, 36 and 48 h after injection. In 22 patients (16 of 38 injected with intact antibodies, 5 of 13 with F(ab')2 fragments and 1 of 2 with Fab' fragments), an increased concentration of 131I radioactivity corresponding to the previously known tumor location was detected by photoscanning 36-48 h after injection. Blood pool and secreted radioactivity was determined in all patients by injecting 15 min before scanning, [99mTc]-labeled normal serum albumin and free 99mTc04-. The computerized subtraction of 99mTc from 131I radioactivity enhanced the definition of tumor localization in the 22 positive patients. However, in spite of the computerized subtraction, interpretation of the scans remained doubtful for 12 patients and was entirely negative for 19 additional patients. In order to provide a more objective evaluation for the specificity of the tumor localization of antibodies, 14 patients scheduled for tumor resection were injected simultaneously with [131I]-labeled antibodies or fragments and with [125I]-labeled normal goat IgG or fragments. After surgery, the radioactivity of the two isotopes present either in tumor or adjacent normal tissues was measured in a dual channel scintillation counter. The results showed that the antibodies or their fragments were 2-4 times more concentrated in the tumor than in the normal tissues. In addition, it was shown that the injected antibodies formed immune complexes with circulating CEA and that the amount of immune complexes detectable in serum was roughly proportional to the level of circulating CEA.

Bone properties and skeletal maturity in adolescent males, as assessed by quantitative ultrasound

ABSTRACT Background: Previous studies have implied that weight-bearing, intense and prolonged physical activities optimize bone accretion during the grow^ing years. The majority of past inquiries have used dual-energy X-ray absorptiometry (DXA) to examine bone strength and hand-wrist radiography to determine skeletal maturity in children. Recently, quantitative ultrasound (QUS) technologies have been developed to examine bone properties and skeletal maturity in a safe, noninvasive and cost-effective manner. Objective: The purpose of this study was to compare bone properties and skeletal maturity in competitive male child and adolescent athletes with minimallyactive, age-matched controls, using QUS technology. >. Methods: In total, 224 males were included in the study. The 115 pre-pubertal boys aged 10-12 years consisted of control, minimally-active children (n=34), soccer players (n=26), gymnasts (n=25) and hockey players (n=30). In addition, the 109 late-pubertal boys aged 14-16 years consisted of control, minimally-active adolescents (n=31), soccer players (n=30), gymnasts (n=17) and hockey players (n=31). The athletic groups were elite level players that predominantly trained year-round. Physical activity, nutrition and sports participation were assessed with various questionnaires. Anthropometries, such as height, weight and relative body fat percentage (BF%) were assessed using standard measures. Skeletal strength and age were evaluated using bone QUS. Lastly, salivary testosterone (sT) concentration was measured using Radioimmunoassay (RIA). Results: Within each age group, there were no significant differences between the activity groups in age and pubertal stage. An age effect was apparent in all variables, as expected. A sport effect was noted in all physical characteristics: the child and adolescent gymnasts were shorter and lighter than other sports groups. Adiposity was greater in the controls and in the hockey players. All child subjects were pubertal stage (fanner) I or II, while adolescent subjects were pubertal stage IV or V. There were no differences in daily energy and mineral intakes between sports groups. In both age groups, gymnasts had a higher training volume than other athletic groups. Bone speed of sound (50s) was higher in adolescents compared with the children. Gymnasts had signifieantly higher radial 50S than controls, hockey and soccer players in both age cohorts. Hockey athletes also had higher radial 50S than controls and soccer players in the child and adolescent groups, respectiyely. Child gymnasts and soccer players had greater tibial 50S compared with the hockey players and control groups. Likewise, adolescent gymnasts and soccer players had higher tibial SoS compared with the control group. No interaction was apparent between age and type of activity in any of the bone measures. » Lastly, maturity as assessed by sT and secondary sex characteristics (Tanner stage) was not different between sports group within each age group. Despite the similarity in chronological age, androgen levels and sexual maturity, differences between activity groups were noted in skeletal maturity. In the younger group, hockey players had the highest bone age while the soccer players had the lowest bone age. In the adolescent group, gymnasts and hockey players were characterized by higher skeletal maturity compared with controls. An interaction between the age and sport type effects was apparent in skeletal maturity, reflecting the fact that among the children, the soccer players were significantly less mature than the rest of the groups, while in the adolescents, the controls were the least skeletally mature. Summary and Conclusions: In summary, radial and tibial SOS are enhanced by the unique loading pattern in each sport (i.e, upper and lower extremities in gymnastics, lower extremities in soccer), with no cumulative effect between childhood and adolescence. That is, the effect of sport participation on bone SOS was apparent already among the young athletes. Enhanced bone properties among athletes of specific sports suggest that participation in these sports can improve bone strength and potential bone health.

Immediate and enduring effects of corticosterone administration during adolescence and adulthood on anxiety and depressive behavior in male rats

Previous research has shown that the stress hormone corticosterone can increase depressive and anxiety-like behavior in rats as well as dampen the HPA response to a novel stressor (Kalynchuk et aI., 2004; Johnson et aI., 2006). Several studies have also shown that adolescence is a period of increased sensitivity to the negative effects of stressors (reviewed in McCormick et aI., 2010), which are often the result of exposure to corticosterone, and yet there is no research to date examining the effects of corticosterone administration during adolescence. The purpose of these experiments is to determine both the immediate and enduring effects of prolonged exposure to corticosterone in adolescence and adulthood on anxiety-like behavior, depressive behavior, and the HPA response. In Experiment 1 adolescent and adult rats were administered an injection of 40 mg/kg of corticosterone or vehicle daily for 16 days. Ha l f of the rats were then tested on the elevated plus maze (EPM) one day after their last injection, and the following day were tested on the forced swim test (FST). After the FST, which is a stressor, blood samples were collected at three time points, and the plasma concentrations of corticosterone were determined using a radioimmunoassay. The remaining rats were left undisturbed for three weeks, and then underwent the same testing as the first group. Corticosterone treatment had little effect on anxiety-like and depressive behavior, but it did alter the HPA response to the FST. In those rats tested soon after the period of injections, corticosterone dampened the HPA response as compared to vehicle treated rats in both adolescent and adult treated rats. For the adolescent treated rats that were tested several weeks later, corticosterone treatment increased HPA response as compared to the vehicle treated rats, but the same was not true for the adult treated rats. I t was hypothesized that the lack of behavioral effects of the corticosterone treatment may be the result of the vehicle injections inducing a stress response and thereby both groups would have similarly altered behavior. In Experiment 2 rats were administered corticosterone dissolved in their drinking water with 2.5% ethanol, or jus t the 2.5% ethanol or plain water, to determine the effects of corticosterone treatment without a stressor present. The regular drinking water was replaced with treated water for 16 days either during adulthood or adolescence, and as before, rats were either tested in the FST one day after the water was removed or three weeks later. Again there was no effect of treatment on depressive behavior. Similar to what was observed in Experiment 1, corticosterone treatment dampened the HPA response to a stressor for the rats tested soon after the treatment period. However, in Experiment 2 there was no effect of treatment on HPA response in those rats tested several weeks after they were treated. These results indicate that corticosterone can have a lasting effect on the HPA when administered in adolescence by injections but not in drinking water, which is likely because of the different schedules of exposure and rates of absorption between the two administration methods.

Le peptide natriurétique auriculaire induit la différenciation cardiaque dans les cellules souches embryonnaires carcinomateuses de souris P19

Traditionnellement associée à la reproduction féminine, l'ocytocine (OT), une hormone peptidique synthétisée par les noyaux paraventriculaire et supraoptique de l'hypothalamus et sécrétée par l'hypophyse postérieure (neurohypophyse), a été récemment revue et a été démontrée avoir plusieurs nouveaux rôles dans le système cardio-vasculaire. En effet, notre laboratoire a montré que l’OT peut induire la différenciation des cellules souches embryonnaires (CSE) en cardiomyocytes (CM) fonctionnels. À l’aide du modèle cellulaire embryonnaire carcinomateux de souris P19, il a été démontré que ce processus survenait suite à la libération de la guanosine monophosphate cyclique (GMPc) dépendante du monoxyde d’azote. De même, il est connu que le peptide natriurétique auriculaire (ANP), un peptide produit, stocké et sécrété par les myocytes cardiaques, peut aussi induire la production du GMPc. De nombreuses études ont démontré que le cœur ayant subi un infarctus pouvait être régénéré à partir d’une population isolée de cellules souches et progénitrices transplantées. Une de ces populations de cellules, fréquemment isolées à partir d'organes provenant d'animaux aux stades de développement embryonnaire et adulte, appelée « Side Population » (SP), sont identifiées par cytométrie en flux (FACS) comme une population de cellules non marquées par le colorant fluorescent Hoechst 33342 (Ho). Les cellules SP expriment des protéines de transport spécifiques, de la famille ATP-binding cassette, qui ont pour rôle de transporter activement le colorant fluorescent Ho de leur cytoplasme. La sous-population de cellules SP isolée du cœur affiche un potentiel de différenciation cardiaque amélioré en réponse à un traitement avec l’OT. Récemment, l'hétérogénéité phénotypique et fonctionnelle des CSE a été mise en évidence, et cela a été corrélé avec la présence de sous-populations cellulaires ressemblant beaucoup aux cellules SP issues du cœur. Puisque l’ANP peut induire la production du GMPc et qu’il a été démontré que la différenciation cardiaque était médiée par la production du GMPc, alors nous émettons l'hypothèse selon laquelle l’ANP pourrait induire la différenciation cardiaque. Étant donné que les CSE sont composés d’un mélange de différents types cellulaires alors nous émettons aussi l’hypothèse selon laquelle l’utilisation d’une sous-population de CSE plus homogène renforcerait le potentiel de différenciation de l'ANP. Méthodes : Les SP ont été isolées des cellules P19 par FACS en utilisant la méthode d’exclusion du colorant fluorescent Ho. Puis, leur phénotype a été caractérisé par immunofluorescence (IF) pour les marqueurs de l’état indifférencié, d’auto-renouvellement et de pluripotence octamer-binding transcription factor 4 (OCT4) et stage-specific embryonic antigen-1 (SSEA1). Ensuite, la dose pharmacologique optimale d’ANP a été déterminée via des tests de cytotoxicité sur des cellules P19 (MTT assay). Pour induire la différenciation en cardiomyocytes, des cellules à l’état de sphéroïdes ont été formées à l’aide de la technique du « Hanging-Drop » sous la stimulation de l’ANP pendant 5 jours. Puis, des cryosections ont été faites dans les sphéroïdes afin de mettre en évidence la présence de marqueurs de cellules cardiaques progénitrices tels que GATA4, Nkx2.5 et un marqueur mitochondrial spécifique Tom22. Ensuite, les cellules SP P19 ont été stimulées dans les sphéroïdes cellulaires par le traitement avec de l'ANP (10-7 M) ou de l’OT (10-7 M), de l’antagoniste spécifique du guanylate cyclase particulé (GCp) A71915 (10-6 M), ainsi que la combinaison des inducteurs OT+ANP, OT+A71915, ANP+A71915. Après la mise en culture, la différenciation en cardiomyocytes a été identifié par l’apparition de colonies de cellules battantes caractéristiques des cellules cardiaques, par la détermination du phénotype cellulaire par IF, et enfin par l’extraction d'ARN et de protéines qui ont été utilisés pour le dosage du GMPc par RIA, l’expression des ARNm par RT-PCR et l’expression des protéines par immunobuvardage de type western. Résultats : Les sphéroïdes obtenus à l’aide de la technique du « Hanging-Drop » ont montré une hausse modeste de l’expression des ARNm suivants : OTR, ANP et GATA4 comparativement aux cellules cultivées en monocouches. Les sphéroïdes induits par l’ANP ont présenté une augmentation significative des facteurs de transcription cardiaque GATA4 et Nkx2.5 ainsi qu’un plus grand nombre de mitochondries caractérisé par une plus grande présence de Tom22. De plus, L’ANP a induit l’apparition de colonies de cellules battantes du jour 7 (stade précoce) au jour 14 (stade mature) de façon presque similaire à l’OT. Cependant, la combinaison de l’ANP avec l’OT n’a pas induit de colonies de cellules battantes suggérant un effet opposé à celui de l’OT. Par IF, nous avons quantifié (nombre de cellules positives) et caractérisé, du jour 6 au jour 14 de différenciation, le phénotype cardiaque de nos cellules en utilisant les marqueurs suivants : Troponine T Cardiaque, ANP, Connexines 40 et 43, l’isoforme ventriculaire de la chaîne légère de myosine (MLC-2v), OTR. Les SP différenciées sous la stimulation de l’ANP ont montré une augmentation significative du GMPc intracellulaire comparé aux cellules non différenciées. À notre grande surprise, l’antagoniste A71915 a induit une plus grande apparition de colonies de cellules battantes comparativement à l’OT et l’ANP à un jour précoce de différenciation cardiaque et l’ajout de l’OT ou de l’ANP a potentialisé ses effets, augmentant encore plus la proportion de colonies de cellules battantes. De plus, la taille des colonies de cellules battantes était encore plus importante que sous la simple stimulation de l’OT ou de l’ANP. Les analyses radioimmunologiques dans les cellules SP P19 stimulés avec l’ANP, A71915 et la combinaison des deux pendant 15min, 30min et 60min a montré que l’ANP stimule significativement la production du GMPc, cependant A71915 n’abolit pas les effets de l’ANP et celui-ci au contraire stimule la production du GMPc via des effets agonistes partiels. Conclusion : Nos résultats démontrent d’une part que l’ANP induit la différenciation des cellules SP P19 en CM fonctionnels. D’autre part, il semblerait que la voie de signalisation NPRA-B/GCp/GMPc soit impliquée dans le mécanisme de différenciation cardiaque puisque l’abolition du GMPc médiée par le GCp potentialise la différenciation cardiaque et il semblerait que cette voie de signalisation soit additive de la voie de signalisation induite par l’OT, NO/GCs/GMPc, puisque l’ajout de l’OT à l’antagoniste A71915 stimule plus fortement la différenciation cardiaque que l’OT ou l’A71915 seuls. Cela suggère que l’effet thérapeutique des peptides natriurétiques observé dans la défaillance cardiaque ainsi que les propriétés vasodilatatrices de certains antagonistes des récepteurs peptidiques natriurétiques inclus la stimulation de la différenciation des cellules souches en cardiomyocytes. Cela laisse donc à penser que les peptides natriurétiques ou les antagonistes des récepteurs peptidiques natriurétiques pourraient être une alternative très intéressante dans la thérapie cellulaire visant à induire la régénération cardiovasculaire.

Mild hypothermia prevents brain edema and attenuates up-regulation of the astrocytic benzodiazepine receptor in experimental acute liver failure.

BACKGROUND/AIMS: Mild hypothermia has proven useful in the clinical management of patients with acute liver failure. Acute liver failure in experimental animals results in alterations in the expression of genes coding for astrocytic proteins including the "peripheral-type" (astrocytic) benzodiazepine receptor (PTBR), a mitochondrial complex associated with neurosteroid synthesis. To gain further insight into the mechanisms whereby hypothermia attenuates the neurological complications of acute liver failure, we investigated PTBR expression in the brains of hepatic devascularized rats under normothermic (37 degrees C) and hypothermic (35 degrees C) conditions. METHODS: PTBR mRNA was measured using semi-quantitative RT-PCR in cerebral cortical extracts and densities of PTBR sites were measured by quantitative receptor autoradiagraphy. Brain pregnenolone content was measured by radioimmunoassay. RESULTS: At coma stages of encephalopathy, animals with acute liver failure manifested a significant increase of PTBR mRNA levels. Brain pregnenolone content and [(3)H]PK 11195 binding site densities were concomitantly increased. Mild hypothermia prevented brain edema and significantly attenuated the increased receptor expression and pregnenolone content. CONCLUSIONS: These findings suggest that an attenuation of PTBR up-regulation resulting in the prevention of increased brain neurosteroid content represents one of the mechanisms by which mild hypothermia exerts its protective effects in ALF.

Glutamate Receptor Gene Expression: IP3, cAMP, cGMP Functional Regulation in Hypoglycaemic and Diabetic Rats

Department of Biotechnology, Cochin University of Science and Technology

Differential effects of two fermentable carbohydrates on central appetite regulation and body composition

BACKGROUND: Obesity is rising at an alarming rate globally. Different fermentable carbohydrates have been shown to reduce obesity. The aim of the present study was to investigate if two different fermentable carbohydrates (inulin and b-glucan) exert similar effects on body composition and central appetite regulation in high fat fed mice. METHODOLOGY/PRINCIPAL FINDINGS: Thirty six C57BL/6 male mice were randomized and maintained for 8 weeks on a high fat diet containing 0% (w/w) fermentable carbohydrate, 10% (w/w) inulin or 10% (w/w) b-glucan individually. Fecal and cecal microbial changes were measured using fluorescent in situ hybridization, fecal metabolic profiling was obtained by proton nuclear magnetic resonance (1H NMR), colonic short chain fatty acids were measured by gas chromatography, body composition and hypothalamic neuronal activation were measured using magnetic resonance imaging (MRI) and manganese enhanced MRI (MEMRI), respectively, PYY (peptide YY) concentration was determined by radioimmunoassay, adipocyte cell size and number were also measured. Both inulin and b-glucan fed groups revealed significantly lower cumulative body weight gain compared with high fat controls. Energy intake was significantly lower in b-glucan than inulin fed mice, with the latter having the greatest effect on total adipose tissue content. Both groups also showed an increase in the numbers of Bifidobacterium and Lactobacillus-Enterococcus in cecal contents as well as feces. b- glucan appeared to have marked effects on suppressing MEMRI associated neuronal signals in the arcuate nucleus, ventromedial hypothalamus, paraventricular nucleus, periventricular nucleus and the nucleus of the tractus solitarius, suggesting a satiated state. CONCLUSIONS/SIGNIFICANCE: Although both fermentable carbohydrates are protective against increased body weight gain, the lower body fat content induced by inulin may be metabolically advantageous. b-glucan appears to suppress neuronal activity in the hypothalamic appetite centers. Differential effects of fermentable carbohydrates open new possibilities for nutritionally targeting appetite regulation and body composition.

A novel association of a polymorphism in the first intron of adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia in Asian Indians

Adiponectin is an adipose tissue specific protein that is decreased in subjects with obesity and type 2 diabetes. The objective of the present study was to examine whether variants in the regulatory regions of the adiponectin gene contribute to type 2 diabetes in Asian Indians. The study comprised of 2,000 normal glucose tolerant (NGT) and 2,000 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. We identified two proximal promoter SNPs (-11377C-->G and -11282T-->C), one intronic SNP (+10211T-->G) and one exonic SNP (+45T-->G) by SSCP and direct sequencing in a pilot study (n = 500). The +10211T-->G SNP alone was genotyped using PCR-RFLP in 4,000 study subjects. Logistic regression analysis revealed that subjects with TG genotype of +10211T-->G had significantly higher risk for diabetes compared to TT genotype [Odds ratio 1.28; 95% Confidence Interval (CI) 1.07-1.54; P = 0.008]. However, no association with diabetes was observed with GG genotype (P = 0.22). Stratification of the study subjects based on BMI showed that the odds ratio for obesity for the TG genotype was 1.53 (95%CI 1.3-1.8; P < 10(-7)) and that for GG genotype, 2.10 (95% CI 1.3-3.3; P = 0.002). Among NGT subjects, the mean serum adiponectin levels were significantly lower among the GG (P = 0.007) and TG (P = 0.001) genotypes compared to TT genotype. Among Asian Indians there is an association of +10211T-->G polymorphism in the first intron of the adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia.

Lack of association between serum adiponectin levels and the Pro12Ala polymorphism in Asian Indians

AIMS: The aim of the study was to investigate the association of serum adiponectin levels with the Pro12Ala polymorphism of the peroxisome proliferator activated receptor-gamma (PPARG) gene in Asian Indians. METHODS: We selected 400 diabetic subjects, 200 with the Pro12Pro genotype (100 male and 100 female) and 200 with the Pro12Ala genotype (100 male and 100 female) and 400 age- and sex-matched normal glucose tolerance subjects with similar genotype profiles from the Chennai Urban Rural Epidemiology Study. Fasting serum adiponection levels were measured using radioimmunoassay. The Pro12Ala polymorphism was genotyped by PCR-restriction fragment length polymorphism using BstUI. RESULTS: All clinical and biochemical parameters were similar in the subjects with the Pro12Pro and Pro12Ala genotypes. There was no significant difference in serum adiponectin values between subjects with the Pro12Pro and Pro12Ala genotypes (males 5.4 vs. 5.8 microg/ml, P = 0.546; females 6.9 vs. 7.2 microg/ml, P = 0.748). Adiponectin values did not differ among these two genotypes even when categorized based on their diabetes status (normal glucose tolerance Pro12Pro 7.9 vs. Pro12Ala 7.7 microg/ml, P = 0.994; diabetes Pro12Pro 4.7 vs. Pro12Ala 5.4 microg/ml, P = 0.622). CONCLUSION: The Pro12Ala polymorphism of the PPARG gene is not associated with serum adiponectin levels in Asian Indians.

Involvement of Phosphatidylinositol-3 Kinase/AKT/PKC zeta/lambda Pathway in the Effect of Palmitate on Glucose-Induced Insulin Secretion

Objectives: In the present study, a novel pathway by which palmilate potentiates glucose-induced insulin secretion by pancreatic beta cells was investigated. Methods: Groups of freshly isolated islets were incubated in 10 mM glucose with palmitate, LY294002, wortmannin, and fumonism B I for measurement of insulin secretion by radioimmunoassay (RIA). Also, phosphorylation and content of AKT and PKC proteins were evaluated by immunoblotting. Results: Glucose plus palmitate and glucose plus LY294002 or wortmannin (PI3K inhibitors) increased glucose-induced insulin secretion by isolated pancreatic islets. Glucose at 10 mM induced AKT and PKC zeta/lambda phosphorylation. Palmitate (0.1 mM) abolished glucose stimulation of AKT and PKC zeta/lambda phosphorylation possibly through PI3K inhibition because both LY294002 (50 mu M) and wortmannin (100 nM) caused the same effect. The inhibitory effect of palmitate on glucose-induced AKT and PKC zeta/lambda phosphorylation and the stimulatory effect of palmitate on glucose-induced insulin secretion were not observed in the presence of fumonisin B1, all inhibitor of ceramide synthesis. Conclusions: These findings support the proposition that palmilate increases insulin release in the presence of 10 mM glucose by inhibiting PI3K activity through a mechanism that involves ceramide synthesis.