Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes

Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1alpha (HIF-1alpha), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H2O2)-induced dysregulation of adiponectin and PAI-1 production. H2O2 treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer binding protein (C/EBPalpha), but had no effect on HIF-1alpha, whereas hypoxia stabilized HIF-1alpha and decreased expression of C/EBPalpha, but not PPARgamma. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases.     

Aldosterone perturbs adiponectin and PAI-1 expression and secretion in 3T3-L1 adipocytes

Aldosterone is considered as a new cardiovascular risk factor that plays an important role in metabolic syndrome; however, the underlying mechanism of these effects is not clear. Hypoadiponectinemia and elevated circulating concentration of plasminogen activator inhibitor-1 (PAI-1) are causally associated with obesity-related insulin resistance and cardiovascular disease. The aim of the present study is to investigate the effect of aldosterone on the production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Northern and Western blot analyses revealed that aldosterone treatment inhibited adiponectin mRNA expression and secretion and simultaneously enhanced PAI-1 mRNA expression and secretion in a time- and dose-dependent manner. Rosiglitazone did not prevent aldosterone's effect on adiponectin or PAI-1 expression. In contrast, tumor necrosis factor (TNF)-α produced dramatic synergistic effects on adiponectin and PAI-1 expression when added together with aldosterone. Furthermore, the effects of aldosterone on adiponectin and PAI-1 expression appear to be mediated through glucocorticoid receptor (GR) but not mineralocorticoid receptor (MR). These results suggest that the effects of aldosterone on adiponectin and PAI-1 production are one of the underlying mechanisms linking it to insulin resistance, metabolic syndrome and cardiovascular disease.     

Production of angiotensin II receptors type one (AT1) and type two (AT2) during the differentiation of 3T3-L1 preadipocytes.

During their development from progenitor cells, adipocytes not only express enzymatic activities necessary for the storage of triglycerides, but also achieve the capability to produce a number of endocrine factors such as leptin, tumor necrosis factor alpha (TNFalpha), complement factors, adiponectin/adipoQ, plasminogen activator inhibitor-1 (PAI-1), angiotensin II and others. Angiotensin II is produced from angiotensinogen by the proteolytic action of renin and angiotensin-converting enzyme; and several data point to the existence of a complete local renin-angiotensin system in adipose tissue, including angiotensin II receptors. In this study, we directly monitored the production of angiotensin II type one receptor (AT1) and angiotensin II type two receptor (AT2) proteins during the adipose conversion of murine 3T3-L1 preadipocytes by immunodetection with specific antibodies. AT1 receptors could be detected throughout the whole differentiation period. The strong AT2 signal in preadipocytes however was completely lost during the course of differentiation, which suggests that expression of AT2 receptors is inversely correlated to the adipose conversion program.     

The alteration of 5-HT(2A) and 5-HT(2C) receptors is involved in neuronal apoptosis of goldfish cerebellum following traumatic experience

5-HT receptor changes remain controversial in posttraumatic stress disorder (PTSD) models. This study looks at the relationship between traumatic injuries and the alterations in 5-HT(2A) and 5-HT(2C) receptors in the goldfish brain. The effect of treatment with doxepin and fluoxetine, known to be selective serotonin reuptake inhibitor (SSRI) antidepressants, on 5-HT receptor expression in goldfish with fin ablation was also investigated. We demonstrated that fin ablation induced anxiety-like behavioural alterations and significant up-regulation of c-fos expression in goldfish cerebellum. The behavioural alterations correlated well with an increased expression of 5-HT(2A) receptors in the cerebellum of the fish with traumatic injury. An increase in the number of apoptotic cells and a higher caspase-8 protein level was present in the brains of goldfish with fin ablation compared to the control. Our findings suggest that neuronal apoptosis occured in the cerebellum as a result of fin ablation and may be related to the alterations of 5-HT(2A) and 5-HT(2C) levels and that the beneficial clinical effects of doxepin/fluoxetine treatment are due to the down-regulation of 5-HT(2A) and up-regulation of 5-HT(2C) receptors in the brain.     

Expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture: integrated response to TNF-alpha

The expression profile of a series of adipokine genes linked to inflammation has been examined by quantitative PCR during the differentiation of human preadipocytes to adipocytes in primary culture, together with the integrated effects of TNF-alpha on the expression of these adipokines in the differentiated adipocytes. Expression of the genes encoding adiponectin, leptin, and haptoglobin was highly differentiation dependent, the mRNA being undetectable predifferentiation with the level peaking 9-15 days postdifferentiation. Although angiotensinogen (AGT) and monocyte chemoattractant protein-1 (MCP-1) were both expressed before differentiation, the mRNA level increased markedly on differentiation. The expression of nerve growth factor (NGF) and plasminogen activator inhibitor-1 (PAI-1) fell after differentiation, whereas that of TNF-alpha and IL-6 changed little. Measurement of adiponectin, leptin, MCP-1, and NGF in the medium by ELISA showed that the protein secretion pattern paralleled cellular mRNA levels. Treatment of differentiated human adipocytes with TNF-alpha (5 or 100 ng/ml for 24 h) significantly decreased the level of adiponectin, AGT, and haptoglobin mRNA (by 2- to 4-fold), whereas that of leptin and PAI-1 was unchanged. In contrast, TNF-alpha induced substantial increases in IL-6, TNF-alpha, metallothionein, MCP-1, and NGF mRNAs, the largest increase being with MCP-1 (14.5-fold). MCP-1 and NGF secretion increased 8- to 10-fold with TNF-alpha, whereas leptin and adiponectin did not change. These results demonstrate that there are major quantitative changes in adipokine gene expression during differentiation of human adipocytes and that TNF-alpha has a pleiotropic effect on inflammation-related adipokine production, the synthesis of MCP-1 and NGF being highly induced by the cytokine.     

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.     

Quantitative assay of 5-HT(1A) serotonin receptor gene expression in the brain

Serotonin 5-HT(1A) receptors participate in the regulation of many kinds of behavior and are implicated in the mechanism of action of anxiolitics and antidepressants. The investigation of 5-HT(1A) receptor gene expression is complicated by low concentration of the receptor mRNA. Our method of quantification of the receptor gene expression in brain structures includes estimation of the concentration of genomic DNA contamination, the number of cDNA copies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)--one of the "housekeeping genes", and the number of cDNA copies of 5-HT(1A) receptor in the sample. To evaluate the number of cDNA copies of the receptor and GAPDH, the fluorescence intensity of PCR-product was calibrated using genomic DNA-standard of a known concentration. The intensity of 5-HT(1A) receptor gene expression was corrected by genomic DNA contamination and was evaluated as a number of copies of 5-HT(1A) receptor cDNA per 100 copies of GAPDH cDNA. Using this method an increase of 5-HT(1A) receptor gene expression in the frontal cortex and amygdala in monoamine oxidase A knockout mice was shown.     

Effect of Mg2+ on guanine nucleotide sensitivity of ligand binding to serotonin1A receptors from bovine hippocampus

The serotonin1A (5-HT1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein coupled receptors (GPCRs). We report here that guanine nucleotide sensitivity of agonist binding to hippocampal 5-HT1A receptors is dependent on the concentration of Mg2+. Our results show that agonist binding to 5-HT1A receptors is relatively insensitive to guanine nucleotides in the absence of Mg2+. In contrast to this, the specific antagonist binding is insensitive to guanine nucleotides, even in the presence of Mg2+. These results point out the requirement of an optimal concentration of Mg2+ which could be used in assays toward determining guanine nucleotide sensitivity of ligand binding to GPCRs such as the 5-HT1A receptor. Our results provide novel insight into the requirement and concentration dependence of Mg2+ in relation to guanine nucleotide sensitivity for the 5-HT1A receptor in particular, and GPCRs in general.     

Proterguride, a highly potent dopamine receptor agonist promising for transdermal administration in Parkinson's disease: interactions with alpha(1)-, 5-HT(2)- and H(1)-receptors

Dopamine receptor agonists play an important role in the treatment of Parkinson's disease and hyperprolactinemic conditions. Proterguride (n-propyldihydrolisuride) was already reported to be a highly potent dopamine receptor agonist, thus its action at different non-dopaminergic monoamine receptors, alpha(1A/1B/1D), 5-HT(2A/2B)- and histamine H(1), was investigated using different functional in vitro assays. The drug behaved as an antagonist at alpha(1)-adrenoceptors without the ability to discriminate between the subtypes (pA(2) values: alpha(1A) 7.31; alpha(1B) 7.37; alpha(1D) 7.35) and showed antagonistic properties at the histamine H(1) receptor. In contrast, at serotonergic receptors (5-HT(2A), 5-HT(2B)) proterguride acted as a partial agonist. The drug stimulated 5-HT(2A) receptors of rat tail artery in lower concentrations than 5-HT itself but failed to evoke comparable efficacy (proterguride: pEC(50) 8.34, E(max) 53% related to the maximum response to 5-HT; 5-HT: pEC(50) 7.03). Agonism at 5-HT(2B) receptors is presently considered to be involved in drug-induced valvular heart disease. Activation of 5-HT(2B) receptors in porcine pulmonary arteries by proterguride (pEC(50) 7.13, E(max) 49%; E(max) (5-HT) 69%), however, occurred at concentrations much higher than plasma concentrations achieving dopaminergic efficacy in humans. The results are discussed focussing on the relevance of action at 5-HT(2B) receptors as well as their significance for a transdermal administration of proterguride. Since it is well accepted that pulsatile dopaminergic stimulation is associated with treatment-related motor complications in the dopaminergic therapy of Parkinson's disease, the transdermal route of administration is of great clinical interest due to the possibility to achieve constant plasma concentrations.     

A 5-HT7 Heteroreceptor-Mediated Inhibition of [3H]Serotonin Release in Raphe Nuclei Slices of the Rat: Evidence for a Serotonergic–Glutamatergic Interaction

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [3H]serotonin ([3H]5-HT), superfused, and the electrically induced efflux of radioactivity was determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated [3H]5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on [3H]5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on [3H]5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked [3H]5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced [3H]5-HT overflow. These findings indicate that 5-CT inhibits [3H]5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited [3H]glutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced [3H]5-HT release.     

Intermittent High Glucose Stimulate MCP-l,IL-18, and PAI-1, but Inhibit Adiponectin Expression and Secretion in Adipocytes Dependent of ROS

Elevated circulating concentrations of interleukin-18 (IL-18), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) and decrease of adiponectin are associated with obesity-related diseases. The mechanism that mediates the aberrant production of the adipokines remains poorly understood. The aim of this study was to investigate the effect of intermittent high glucose on the expression of IL-18, MCP-1, and PAI-1 and adiponectin in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated for 24 h in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l and a daily alternating 5 or 20 mmol/l glucose, with or without the addition of 1.0 mmol/l N-acetylcysteine (NAC). The expression and secretion of IL-18, MCP-1, PAI-1, and adiponectin were determined by real-time RT-PCR and ELISA, respectively. The production of reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine (8-OHdG) were measured. Stable high glucose significantly increased expression and secretion of IL-18, MCP-1, and PAI-1, and reduced adiponectin expression and secretion compared to normal glucose conditions. These effects were significantly greater under intermittent high glucose conditions compared to stable high glucose. The level of ROS and 8-OHdG were significantly elevated under both intermittent and stable high glucose conditions, the effect being greater under intermittent high glucose. The intermittent glucose was more effective in triggering the generation of ROS than stable high glucose. The adding of the NAC, a specific pharmacological inhibitor of ROS, normalized the expression of these adipokines and the levels of ROS and 8-OHdG under both stable and intermittent glucose conditions. Intermittent high glucose induces a greater aberrant production of key adipokines than stable high glucose, and this effect seems to be related to over-production of ROS.     

Dibutyryl-cAMP increases functions of 5-hydroxytryptamine2 receptors, but not of beta 2-adrenergic receptors, in a clonal cell line of rat neurotumor RT4.

A peripheral nervous system cell line RT4-B, established by Imada and Sueoka (Dev. Biol., 66:97-108, 1978), was shown to respond to serotonin [5-hydroxytryptamine (5-HT)] and catecholamines. 5-HT induced a small and transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) in the RT4-B cells. The increase was effectively blocked by 5-HT2 receptor antagonists (spiperone, ritanserin and mianserin), but not by a 5-HT3 receptor antagonist (MDL72222), or a alpha 1-adrenergic receptor antagonist (prazosin), indicating that RT4-B cells express 5-HT2 receptors. On the other hand, catecholamines increased cyclic AMP production by RT4-B. The order of potency for stimulating cyclic AMP synthesis was isoproterenol greater than epinephrine much greater than norepinephrine much greater than dopamine, and the stimulation was effectively inhibited by the nonselective beta-adrenergic receptor antagonist propranolol, but not by the beta 1-adrenergic receptor antagonist atenolol, suggesting that RT4-B cells express beta 2-adrenergic receptors. The differentiating agent N6,2'-O-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) enhanced the 5-HT-induced [Ca2+]i increase, but not the catecholamine-induced cyclic AMP production. The increase in the 5-HT response paralleled the increase in the density of 5-HT2 receptors. n-Butyric acid (2 mM) and 8-bromoadenosine 3',5'-monophosphate (1 mM) also increased the 5-HT response, and the sum of these increases was nearly equal to that induced by dibutyryl-cAMP. These results indicate that RT4-B is a novel model cell line for the study of 5-HT2 and beta 2-adrenergic receptors and their second messenger responses and for the analysis of the mechanisms how 5-HT2 receptor gene expression is controlled.     

Effects of adrenal hormones on the expression of adiponectin and adiponectin receptors in adipose tissue, muscle and liver

Background

Adiponectin, an insulin-sensitive hormone that is primarily synthesized in adipose tissue, exerts its effects by binding to two receptors, adipoR1 and adipoR2. Little is known regarding the effects of glucocorticoids on the expression of adiponectin receptors.

Methods

Male Wistar rats were bilaterally adrenalectomized and treated with dexamethasone (0.2 mg/100 g) twice daily for 3 days. To analyze the potential effects of glucocorticoids, rats received two daily injections of the glucocorticoid receptor antagonist (RU-486, 5.0 mg) over the course of 3 days. Additionally, 3T3-L1 adipocytes and C2C12 myotubes were treated with dexamethasone, adrenaline or RU-486. The gene expression of adiponectin, adipoR1 and adipoR2 was determined by real-time PCR, and protein secretion was examined by Western blotting using lysates from retroperitoneal, epididymal and subcutaneous adipose tissue depots, liver and muscle.

Results

In rats, excess glucocorticoids increased the levels of insulin in serum and decreased serum adiponectin concentrations, whereas adrenalectomy decreased the mRNA expression of adiponectin (3-fold) and adipoR2 (7-fold) in epididymal adipose tissue and increased adipoR2 gene expression in muscle (3-fold) compared to control group sham-operated. Dexamethasone treatment did not reverse the effects of adrenalectomy, and glucocorticoid receptor blockade did not reproduce the effects of adrenalectomy. In 3T3-L1 adipocytes, dexamethasone and adrenaline both increased adipoR2 mRNA levels, but RU-486 reduced adipoR2 gene expression in vitro.

Conclusion

Dexamethasone treatment induces a state of insulin resistance but does not affect adiponectin receptor expression in adipose tissue. However, the effects of catecholamines on insulin resistance may be due to their effects on adipoR2.     

Effects of the new C1q/TNF-related protein (CTRP-3) "cartonectin" on the adipocytic secretion of adipokines

Background: Cartonectin (collagenous repeat‐containing sequence of 26‐kDa protein; CORS‐26) was described as a new adipokine of the C1q/TNF molecular superfamily C1q/TNF‐related protein‐3 (CTRP‐3), secreted by the adipocytes of mice and humans. The receptor and function of cartonectin are unknown and the recombinant protein is not commercially available. Objective: To investigate the effects of recombinant cartonectin on the secretion of adipokines such as adiponectin, leptin, and resistin from adipocytes of human and murine origin. The effect of the BMI of the adipocyte donor was also investigated. Methods and Procedures: Human adipocytes from pooled lean and preobese healthy individuals and murine 3T3‐L1 adipocytes were used for stimulation experiments. Recombinant cartonectin was expressed in insect H5 cells. Adipokine secretion was measured using enzyme‐linked immunosorbent assay. In addition, western blot analysis and luciferase reporter gene assays were employed. Results: Cartonectin (1, 10, 50, and 250 ng/ml) in higher doses stimulates the secretion of adiponectin and resistin from murine adipocytes. This effect is not caused by an induction of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) protein expression, as confirmed by western blot analysis. Also, luciferase reporter gene assay revealed that cartonectin failed to induce luciferase activity at the peroxisome proliferator‐activated receptor responsive element site containing the adiponectin/luciferase promoter fragment. Human adipocytes from lean individuals secrete higher amounts of adiponectin and leptin when compared with adipocytes of individuals with a preobesity BMI (25–30 kg/m²). Cartonectin failed to stimulate adiponectin or leptin secretion from human adipocytes, irrespective of the BMI value. Discussion: Cartonectin is a new adipokine that differentially regulates the secretion of classical adipokines, with marked differences between the human and the murine systems. These effects are species‐dependent, while basal adipokine secretion is influenced by the BMI.