Psychosocial profile of the metabolically healthy but obese postmenopausal woman.
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Body Mass Index↗
Biomedical subjects
E Doucet
Publications and source records attributed to E Doucet.
Greater than predicted decrease in resting energy expenditure with age: cross-sectional and longitudinal evidence.
OBJECTIVE: To determine whether the age-related decrease in resting energy expenditure (REE) is explained by variations in body composition. DESIGN: In Study 1, adult subjects (20-70 years) from the Quebec Family Study were classified into five different age groups. Body composition was measured by hydrodensitometry to determine fat mass and fat-free mass as predictors of REE. In the youngest group of individuals these predictors were used to plot a reference regression that was then used to predict REE in the other age groups. In Study 2, this issue was investigated in a longitudinal design (6-year follow-up). Subjects were subdivided into three groups and a reference regression was plotted at the beginning of the follow-up and was then used to predict REE 6 years later in the three age groups. SUBJECTS: In Study 1, 627 adults (288 men and 339 women), aged between 20 and 70 years. In Study 2, 191 adults (93 men and 98 women). RESULTS: In Study 1, measured REE was 329, 302, 528 and 636 kJ/day (P < 0.0001) below predicted REE at 34, 44, 54 and 64 years, respectively. In Study 2 the most marked deviation from predicted REE in response to the 6-year follow-up in men was observed in young adults (-548 kJ/day, P < 0.001) while in women, the largest deviation occurred later in life (-720 kj/day, P < 0.001). CONCLUSION: Aging is accompanied by a decrease in REE that is significantly greater than what is predicted by variations in body composition. This decrease may reach a mean level of about 500-800 kj/day.
Absorptiometry, Photon↗
Validity of foot-to-foot bioelectrical impedance analysis in overweight and obese children and parents.
AIM: The aim of this study was to evaluate the validity of foot-to-foot bioelectrical impedance analysis (FF-BIA) in the measurement of body composition in overweight and obese children and their parents by comparison to dual-energy X-ray absorptiometry (DXA). METHODS: Seventeen, 7-12 year old overweight and obese children (6 boys, 11 girls) and 17 parents (5 fathers, 12 mothers) were evaluated for body composition with FF-BIA and DXA. Measures of percent body fat (PBF), fat mass (FM) and fat free mass (FFM) derived from FF-BIA and DXA were compared. Measures of validity were determined by Pearson correlations between FF-BIA and DXA, paired t-tests to assess mean differences, as well as biases and limits of agreement using the Bland Altman tests. RESULTS: FF-BIA produced estimates of body composition that were highly correlated with DXA in overweight and obese children and parents. For children, the correlations for PBF, FM, and FFM were 0.85, 0.97, and 0.94, respectively. For parents, the correlations for PBF, FM, and FFM were 0.92, 0.97, and 0.91, respectively. However, mean differences between FF-BIA and DXA were significant in children but not in parents. Bland-Altman tests of agreement showed moderate to large within-subject differences in body composition variables between FF-BIA and DXA. CONCLUSIONS: FF-BIA is strongly related to DXA in the measurement of body composition in both overweight and obese preadolescent children and parents, but the two measures may not be used interchangeably. Although FF-BIA may lack the precision to assess small changes in body composition in overweight and obese individuals, it is appropriate for epidemiological use.
Absorptiometry, Photon↗
Is the insulin resistance syndrome the price to be paid to achieve body weight stability?
The insulin resistance syndrome represents a metabolic state in which hyperinsulinemia and/or insulin resistance constitute the platform underlying the development of metabolic complications and related diseases such as diabetes and coronary heart disease. As described in this paper, these insulin-related changes are also involved in the regulation of energy balance and contribute to the recovery of body weight stability in a context of long-term positive energy balance. Under conditions of negative energy balance such as obesity treatment, this connection has a high clinical relevance. In this regard, relevant literature as well as the reanalysis of previously published data suggest that the beneficial effect of weight loss on insulin-related changes in glycemia cannot be disassociated from the relationship between changes in plasma insulin and those in resting energy expenditure. In clinical terms, this suggests that the beneficial effect of weight loss on components of the insulin resistance syndrome could be related to the development of a state of physiological vulnerability that complicates the control of body weight. This poses a major challenge to health professionals who then have to manage obesity treatment as the search for a compromise between the beneficial and potentially detrimental effects of weight loss on insulinemia and insulin sensitivity. This also reinforces the relevance to adhere to healthy diet and physical activity habits in order to maintain body weight stability rather than relying on the overuse of regulatory systems soliciting the effects of hyperinsulinemia on the control of energy intake and expenditure.
Energy Metabolism↗
Estimation of daily energy needs with the FAO/WHO/UNU 1985 procedures in adults: comparison to whole-body indirect calorimetry measurements.
OBJECTIVES: To measure daily energy expenditure (DEE) with indirect calorimetric facilities in sedentary and active subjects. To estimate daily energy needs with the FAO/WHO/UNU (1985) procedures (EDEE) and estimated energy requirement (EER) with the dietary reference intakes 2002 (DRI) in healthy adults with sedentary or high-activity conditions. To compare estimated daily energy needs with their measured values. DESIGN: Two groups of healthy subjects were tested under sedentary or high-activity conditions. In both groups, resting energy expenditure was measured after a 12-h overnight fast. DEE and basal metabolic rate (BMR) values were also measured with indirect calorimetry and compared to the relevant predicted values. Physical activity level and BMR were also estimated. SUBJECTS: A total of 45 sedentary (26 men and 19 women) and 69 active subjects (43 men and 26 women) aged 18-30 and 30-60 y. RESULTS: Measured daily energy expenditure (MDEE) was significantly lower than EDEE in sedentary men and women and in active men for the two age groups considered (P<0.05). EER was significantly lower than EDEE in both sedentary and active subjects of each subgroup (P<0.05). CONCLUSIONS: The FAO/WHO/UNU (1985) procedures may overestimate daily energy needs, particularly in sedentary individuals. However, DRI (2002) are probably more adapted to estimate real daily energy needs in sedentary and active subjects in comparison to the FAO/WHO/UNU (1985) procedures.
Adolescent↗
Thermogenesis and weight loss in obese individuals: a primary association with organochlorine pollution.
The main objective of this study was to investigate the potential impact of body organochlorine (OC) pollution on the adaptive change in thermogenesis induced by body weight loss. Fat mass (FM), fat-free mass (FFM), and sleeping metabolic rate (SMR) were measured in obese individuals before and after a weight-reducing program. The measured values of SMR were then compared to those predicted from a reference equation established from FM and FFM in control subjects. Plasma OC, leptin, total tri-iodothyronine, and free thyroxine concentrations were also measured in obese subjects before and after weight loss. After weight loss, the measured decrease in SMR was greater than that predicted by changes in FM and FFM. Increased plasma OC concentration was the factor explaining the greatest proportion of the difference between predicted and measured SMR changes induced by body weight loss. OC pollution seems to be a new factor affecting the control of thermogenesis in some obese individuals experiencing body weight loss.
Adult↗
Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.
The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.
Animals↗
Reduction of visceral adipose tissue during weight loss.
OBJECTIVE: The aims of the present study were to retrospectively: (1) compare how weight loss affects the reduction of adipose tissue from three different sites between men and women; and (2) to verify whether gender differences in the reduction of adipose tissue are influenced by changes in fat mass (FM) and initial levels of fat in different compartments. DESIGN: Double-blind randomized treatment with fenfluramine once daily coupled to a non-macronutrient specific energy restriction. SUBJECTS: Seventeen obese men (age 43.9+/-1.5 and body mass index (BMI) 34.3+/-0.7) and 17 obese women (age 41.2+/-1.2 and BMI 35.7+/-0.6). INTERVENTIONS: Subjects were given fenfluramine (60 mg) or placebo once daily and were also subjected to a non-macronurient specific energy restriction of -2.9 MJ/day (-700 kcal/day) for 15 weeks. RESULTS: Body weight, FM, fat-free mass (FFM), waist circumference, BMI, as well as visceral (VAT), subcutaneous abdominal (SAT) and thigh (TAT) adipose tissue were all significantly reduced. Men lost significantly more VAT (-41.6%) than SAT (-22.5%), or than TAT (-20.5%) while no site difference in fat loss was observed in women when changes were calculated as a percentage of initial levels. Men lost about twice as much fat from the VAT compartment than did women (P<0.05), even after having considered changes in FM as a potential covariate. In absolute values, TAT was reduced to a lesser extent in men than in women. However, when initial levels of respective fat depots were also taken into account, gender differences in VAT and TAT loss were no longer statistically significant. CONCLUSION: These results suggest that gender differences in VAT reduction during weight loss are independent of changes in FM. However, once initial levels of VAT are also taken into account, gender differences in the reduction of this tissue during weight loss are no longer apparent.
Adipose Tissue↗
Differential subcellular localization of the 5-HT3-As receptor subunit in the rat central nervous system.
Following the cloning and sequencing of the A subunit of the 5-HT3 receptor, two alternatively spliced isoforms, 5-HT3-AS and 5-HT3-AL, have been identified. In order to analyse the distribution of the receptor, a polyclonal antibody has been produced against the short form which is the most abundant in the central nervous system [Doucet et al. (2000) Neuroscience 95, 881-892]. As expected from the recognition of functional 5-HT3 receptors, immunostaining by this anti-5-HT3-R-AS antibody matched the distribution of the high-affinity 5-HT3 binding sites in the rat brain and spinal cord. 5-HT3-AS-like immunoreactivity was detected at low levels in the limbic system, particularly in the amygdala and the hippocampus, and in the frontal, piriform and entorhinal cortices. High levels of immunoreactivity were found in the brainstem, mainly in the nucleus tractus solitarius and the nucleus of the spinal tract of the trigeminal nerve, and in the dorsal horn of the spinal cord. At the ultrastructural level, immunostaining was generally found associated with axons and nerve terminals (70-80%) except in the hippocampus, where labelled dendrites were more abundant (56%). This preferential localization on nerve endings is consistent with the well-documented physiological role of 5-HT3 receptors in the control of neurotransmitter release. However, the different distribution in the hippocampus raises the question of whether differential addressing mechanisms exist for preferentially targeting 5-HT3 receptors to postsynaptic dendritic sites as compared to presynaptic nerve endings, depending on the nature of the neurons bearing these receptors.
Animals↗
Agonist-induced internalization of serotonin-1a receptors in the dorsal raphe nucleus (autoreceptors) but not hippocampus (heteroreceptors).
Serotonin-1A (5-HT(1A)) receptors in the CNS are a major target for psychotropic drugs. In nucleus raphe dorsalis (NRD) and hippocampus (CA3), the selective 5-HT(1A) agonist (+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) reduces the firing activity of serotoninergic (5-HT) and pyramidal neurons, respectively. When located on 5-HT (autoreceptors), but not on non-5-HT (heteroreceptors) neurons, 5-HT(1A) receptors are known to be subject to desensitization. Using quantitative electron microscopy after pre-embedding immunogold labeling with specific antibodies, we examined the subcellular distribution of these receptors after acute administration of 8-OH-DPAT (0.5 mg/kg, i.v.). Silver-intensified immunogold particles associated with the plasma membrane or the cytoplasm were counted in somata and dendrites within the NRD, 15 min, 1 hr and 24 hr after 8-OH-DPAT injection, and in hippocampal dendrites 1 hr after the same treatment. Significant decrease in the density of membrane labeling and concomitant increase of cytoplasmic labeling were demonstrated in the NRD, 15 min and 1 hr after 8-OH-DPAT administration, with a return to baseline level at 24 hr. Internalization was blocked by previous administration of the 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide (WAY 100635), which, by itself, was without apparent effect. In hippocampus (CA3), there were no apparent changes in the distribution of the receptor after 8-OH-DPAT administration. These findings are in line with earlier results showing a desensitization of 5-HT(1A) autoreceptors but not heteroreceptors after treatment with 5-HT(1A) receptor agonist. They suggest that this desensitization is the result of autoreceptor internalization.
8-Hydroxy-2-(di-n-propylamino)tetralin↗
Impact of high-intensity exercise on energy expenditure, lipid oxidation and body fatness.
OBJECTIVE: Two studies were conducted to assess the potential of an increase in exercise intensity to alter energy and lipid metabolism and body fatness under conditions mimicking real life. METHODS: Study 1 was based on the comparison of adiposity markers obtained in 352 male healthy adults who participated in the Québec Family Study who either regularly participated in high-intensity physical activities or did not. Study 2 was designed to determine the effects of high-intensity exercise on post-exercise post-prandial energy and lipid metabolism as well as the contribution of beta-adrenergic stimulation to such differences under a real-life setting. RESULTS: Results from Study 1 showed that men who regularly take part in intense physical activities display lower fat percentage and subcutaneous adiposity than men who never perform such activities, and this was true even if the latter group reported a lower energy intake (917 kJ/day, P<0.05). In Study 2, the high-intensity exercise stimulus produced a greater post-exercise post-prandial oxygen consumption as well as fat oxidation than the resting session, an effect which disappeared with the addition of propranolol. In addition, the increase in post-prandial oxygen consumption observed after the high-intensity exercise session was also significantly greater than that promoted by the low-intensity exercise session. CONCLUSION: These results suggest that high-intensity exercise favors a lesser body fat deposition which might be related to an increase in post-exercise energy metabolism that is mediated by beta-adrenergic stimulation.
Adipose Tissue↗
Obesity treatment with a progressive clinical tri-therapy combining sibutramine and a supervised diet--exercise intervention.
OBJECTIVE: : Sibutramine favors a negative energy balance and also has the potential to increase heart rate and blood pressure. We investigated if a progressive supervised sibutramine--diet--exercise clinical intervention could increase the body weight loss previously reported while minimizing the potential cardiostimulatory effects of this drug. DESIGN AND SUBJECTS: : The tri-therapy intervention was divided into two phases of 6 weeks each in which sibutramine (10 mg) was taken once daily by eight obese men (body mass index (BMI) between 30 and 40 kg/m(2)). Part A consisted of a dietary follow-up with an energy restriction, whereas in part B an aerobic exercise program combined with a low-fat diet was introduced. Systolic (SBP) and diastolic (DBP) blood pressure, resting heart rate (RHR) and body weight were measured every 2 weeks while body density, resting metabolic rate (RMR) and respiratory quotient (RQ) were determined before and after the intervention. RESULTS: : This clinical intervention produced a substantial body weight loss (-10.7 kg, P<0.01) which was about twice as much as other 12-week studies. In part A, both RHR (+4 beats/min) and DBP (+5 mmHg, P<0.01) were increased. However, after part B, RHR (-8 beats/min, P=0.02) and DBP (-3 mmHg, P<0.01) were significantly decreased. RMR was decreased at the end of the program but this effect did not persist after adjustments for fat-free mass. RQ was also reduced (-0.05, P<0.01) following the clinical tri-therapy. CONCLUSION: : In conclusion, these observations suggest that this clinical tri-therapy favored a satisfactory benefit--risk profile since it enhanced weight loss without inducing increases in heart rate and blood pressure or detrimental changes in RMR and substrate oxidation.
Adult↗
Difference in leptin response to a high-fat meal between lean and obese men.
The aim of this study was to compare the leptin responses to a high-fat meal in lean and obese men, and to investigate whether the net leptin response (area under the incremental curve) after the meal was related to the thermic effect of food (TEF). Blood samples were collected after an overnight fast and every 2 h for 8 h after a high-fat breakfast (60 g of fat/m(2) body surface area) in 12 lean and 12 obese men for determination of glucose, insulin and leptin. The TEF was calculated as postprandial energy expenditure minus fasting energy expenditure, as measured by indirect calorimetry. Fasting plasma glucose levels were similar in lean and obese men, and increased in the same way after the meal. Fasting and postprandial plasma insulin concentrations were significantly greater in obese than in lean men (P<0.01 and P<0.05 respectively). Accordingly, obese men showed a significantly higher net insulin response than lean subjects (P<0.001). Fasting plasma leptin levels were greater in obese than in lean men (P<0.001). After the meal, plasma leptin increased significantly in lean men, whereas it decreased in obese men (group by time interaction, P<0.01). The net response of leptin was greater in lean than in obese men, but this did not reach statistical significance (P=0.07). Moreover, the TEF was similar in the two groups. No significant relationship was observed between either the net insulin response or the net leptin response after the high-fat meal and the TEF of lean subjects (-0.05 <r<0.31). In obese men, the net response of insulin was correlated significantly with TEF (r=0.70, P<0.05), whereas the net response of leptin was not (r=-0.40). These results suggest that obesity is related to an impaired regulation of leptin by insulin, since leptin levels increased in lean men but decreased in obese men following a high-fat meal. Moreover, the fact that the postprandial leptin responses of both lean and obese men were not significantly related to the TEF suggests that the ob gene product is probably not acutely involved in the control of this energy expenditure component in humans.
Adult↗
Combined effects of red pepper and caffeine consumption on 24 h energy balance in subjects given free access to foods.
The effects of red pepper and caffeine ingestion on energy and macronutrient balances were examined in eight Caucasian male subjects. All subjects participated in two randomly assigned conditions: control and experimental (red pepper and caffeine). After ingesting a standardized breakfast, subjects ate three meals ad libitum (lunch, dinner and breakfast) and snacks which were served approximately 2 h after the lunch and dinner over a 24 h period. Two appetizers with or without 3 g red pepper) were given before lunch and dinner, and a drink (decaffeinated coffee with or without 200 mg caffeine) was served at all meals and snacks except for the after-dinner snack. It is also important to note that on the experimental day, 8.6 and 7.2 g red pepper were also added to lunch and dinner respectively. Red pepper and caffeine consumption significantly reduced the cumulative ad libitum energy intake and increased energy expenditure. The mean difference in energy balance between both conditions was 4000 kJ/d. Moreover, the power spectral analysis of heart rate suggested that this effect of red pepper was associated with an increase in sympathetic:parasympathetic nervous system activity ratio. These results indicate that the consumption of red pepper and caffeine can induce a considerable change in energy balance when individuals are given free access to foods.
Adult↗
Evidence for the existence of adaptive thermogenesis during weight loss.
The present study was performed to further investigate the adaptive component of thermogenesis that appears during prolonged energy restriction. Fifteen obese men and twenty obese women underwent a 15-week weight-loss programme. During this programme, body weight and composition as well as resting energy expenditure (REE) were measured at baseline, after 2 and 8 weeks of energy restriction (-2929 kJ/d) and drug therapy (or placebo), and finally 2-4 weeks after the end of the 15-week drug therapy and energy restriction intervention, when subjects were weight stable. Regression equations were established in a control population of the same age. These equations were then used to predict REE in obese men and women at baseline, after 2 and 8 weeks, as well as after the completion of the programme. In both men and women body weight and fat mass were significantly reduced in all cases) while fat-free mass remained unchanged throughout the programme. At baseline, REE predicted from the regression equation was not significantly different from the measured REE in men, while in women the measured REE was 13 % greater than predicted. After 2 weeks of energy restriction, measured REE had fallen by 469 and 635 kJ/d more than predicted and this difference reached 963 and 614 kJ/d by week 8 of treatment in men and women respectively. Once body-weight stability was recovered at the end of the programme, changes in REE remained below predicted changes in men (-622 kJ/d). However, in women changes in predicted and measured REE were no longer different at this time, even if the women were maintaining a reduced body weight. In summary, the present results confirm the existence of adaptive thermogenesis and give objective measurements of this component during weight loss in obese men and women, while they also emphasize that in women this component seems to be essentially explained by the energy restriction.
Adaptation, Physiological↗
Elevated intramyocellular lipid concentration in obese subjects is not reduced after diet and exercise training.
To determine the effects of weight loss on intramyocellular energy substrates, vastus lateralis muscle biopsies were taken from six obese subjects (body mass index 34 +/- 5 kg/m(2)) before, after 15 wk of energy restriction (ER; -700 kcal/day), and after a further average 20.7 +/- 1.6 wk of endurance training plus low-fat diet (ET-LFD). Body weight fell from 100 +/- 6 to 89 +/- 6 kg during ER and to 84 +/- 4 kg after ET-LFD. Lipids and glycogen were histochemically measured in type I, IIA, and IIB fibers. Total muscle glycogen content (MGC; per 100 fibers) decreased after ER [from 72 +/- 13 to 55 +/- 8 arbitrary units (AU)]. A similar but not significant decrease was seen in total muscle lipid content (MLC; 14 +/- 5 to 9 +/- 1 AU). After ET-LFD, MGC returned to initial values (74 +/- 8 AU), and MLC approached near-initial values (12 +/- 3 AU). Individual fiber lipid concentration did not change throughout the protocol in all fiber types, whereas glycogen concentration increased after ET-LFD. The training effects of ET-LFD were measured as increasing activities of key mitochondrial enzymes. Although total muscle energy reserves can be reduced after weight loss, their concentration within individual myofibers remains elevated. Weight loss does not appear sufficient to correct the potential detrimental effects of high intracellular lipid concentrations.
Adult↗
Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain.
The 5-HT1A and 5-HT1B receptors of serotonin play important roles as auto- and heteroreceptors controlling the release of serotonin itself and of other neurotransmitters/modulators in the central nervous system (CNS). To determine the precise localization of these receptors, we examined their respective cellular and subcellular distributions in the nucleus raphe dorsalis and hippocampal formation (5-HT1A) and in the globus pallidus and substantia nigra (5-HT1B), using light and electron microscopic immunocytochemistry with specific antibodies. Both immunogold and immunoperoxidase preembedding labelings were achieved. In the nucleus raphe dorsalis, 5-HT1A immunoreactivity was found exclusively on neuronal cell bodies and dendrites, and mostly along extrasynaptic portions of their plasma membrane. After immunogold labeling, the density of membrane-associated 5-HT1A receptors could be estimated to be at least 30-40 times that in the cytoplasm. In the hippocampal formation, the somata as well as dendrites of pyramidal and granule cells displayed 5-HT1A immunoreactivity, which was also prominent on the dendritic spines of pyramidal cells. In both substantia nigra and globus pallidus, 5-HT1B receptors were preferentially associated with the membrane of fine, unmyelinated, preterminal axons, and were not found on axon terminals. A selective localization to the cytoplasm of endothelial cells of microvessels was also observed. Because the 5-HT1A receptors are somatodendritic, they are ideally situated to mediate serotonin effects on neuronal firing, both as auto- and as heteroreceptors. The localization of 5-HT1B receptors to the membrane of preterminal axons suggests that they control transmitter release from nonserotonin as well as serotonin neurons by mediating serotonin effects on axonal conduction. The fact that these two receptor subtypes predominate at extrasynaptic and nonsynaptic sites provides further evidence for diffuse serotonin transmission in the CNS.
Animals↗
Immunolabeling of the rat central nervous system with antibodies partially selective of the short form of the 5-HT3 receptor.
Polyclonal antibodies were raised against a synthetic hexadecapeptide corresponding to the portion of the second intracytoplasmic loop of the short form of the mouse 5-hydroxytryptamine-3A receptor subunit (5-HT3A-S), which differs from the long form (5-HT3A-L) by the removal of six amino acids. Antibodies were detected by enzyme-linked immunosorbent assay as soon as two months after the first injection to rabbits of the peptide coupled to keyhole limpet hemocyanin. Immunoblot detection of fusion proteins comprising glutathione-S-transferase and the second intracellular loop of 5-HT3A-S or 5-HT3A-L, and immunoprecipitation of cloned receptors showed that antibodies exhibited some selectivity for the short variant. Affinity chromatography allowed the purification of selective anti-5-HT3A-S antibodies which yielded a strong positive labeling of plasma membrane, reticulum and Golgi apparatus of COS-7 cells expressing murine 5-HT3A-S. In contrast, COS-7 cells expressing similar levels of 5-HT3A-L exhibited only a very weak labeling. Selectivity was also observed on immunoblots of cloned receptors transiently expressed in COS-7 cells, or stably expressed in CHO cells, both systems showing an immunolabeled component at 53,000-54,000 mol. wt. Immunoautoradiographic labeling of central nervous system sections showed that 5-HT3A-S-like immunoreactivity was found mostly within the nucleus of the solitary tract, the nucleus of the spinal tract of the trigeminal nerve, and the dorsal horn of the the spinal cord in the rat. After unilateral ablation of the nodose ganglion, 5-HT3A-S-like immunoreactivity decreased markedly in the ipsilateral part of the nucleus of the solitary tract, as expected of the presynaptic localization of 5-HT3 receptors. Finally, immunohistochemistry at the light and electron microscope levels revealed that 5-HT3A-S-like immunoreactivity was associated essentially with terminals and axonal profiles. All these results demonstrate that the immunolabeling exhibited by these antibodies is consistent with a specific and partially selective recognition of the short isoform of the 5-HT3A subunit. Because the pattern of immunoautoradiographic labeling matches the distribution previously established with selective radioligands, it can be inferred that these antibodies probably recognized the same fully assembled form of the 5-HT3A-S receptor subunit.
Amino Acid Sequence↗