Louise Sanders

Activators of peroxisome proliferator-activated receptor gamma have depot-specific effects on human preadipocyte differentiation.

Activation of peroxisome proliferator-activated receptor (PPAR) gamma, a nuclear receptor highly expressed in adipocytes, induces the differentiation of murine preadipocyte cell lines. Recently, thiazolidinediones (TZDs), a novel class of insulin-sensitizing compounds effective in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) have been shown to bind to PPARgamma with high affinity. We have examined the effects of these compounds on the differentiation of human preadipocytes derived from subcutaneous (SC) and omental (Om) fat. Assessed by lipid accumulation, glycerol 3-phosphate dehydrogenase activity, and mRNA levels, subcultured preadipocytes isolated from either SC or Om depots did not differentiate in defined serum-free medium. Addition of TZDs (BRL49653 or troglitazone) or 15-deoxyDelta12,14prostaglandin J2 (a natural PPARgamma ligand) enhanced markedly the differentiation of preadipocytes from SC sites, assessed by all three criteria. The rank order of potency of these agents in inducing differentiation matched their ability to activate transcription via human PPARgamma. In contrast, preadipocytes from Om sites in the same individuals were refractory to TZDs, although PPARgamma was expressed at similar levels in both depots. The mechanism of this depot-specific TZD response is unknown. However, given the association between Om adiposity and NIDDM, the site-specific responsiveness of human preadipocytes to TZDs may be involved in the beneficial effects of these compounds on in vivo insulin sensitivity.

Depot- and Sex-Specific Differences in Human Leptin mRNA Expression: Implications for the Control of Regional Fat Distribution

Obese subjects with excess intra-abdominal fat deposition suffer greater adverse metabolic consequences than do similarly overweight subjects with a predominantly subcutaneous distribution of adiposity. Little is known about the factors regulating the regional distribution of body fat. Leptin is a recently characterized protein secreted by adipocytes that appears to provide a long-term hormonal feedback signal regulating fat mass. No systematic evaluation of site-related differences in human adipocyte leptin expression has been reported to date. Levels of leptin mRNA were examined by quantitative reverse transcription-polymerase chain reaction in adipocytes isolated from omental and subcutaneous adipose depots of nonobese and mildly obese individuals undergoing elective surgery. In all individuals studied (n = 24), leptin mRNA levels were higher in subcutaneous than in omental adipocytes (P < 0.0001). In contrast, there were no consistent site-specific differences in the expression of glycerol-3-phosphate dehydrogenase mRNA. The subcutaneous-to-omental ratio of leptin mRNA expression was markedly higher in women (5.5 +/- 1.1-fold) than in men (1.9 +/- 0.2-fold) (P < 0.02). A significant relationship between BMI and leptin mRNA expression was demonstrable in the subcutaneous adipocytes of women (P < 0.006). Thus, leptin mRNA appears to be expressed predominantly by subcutaneous adipocytes, particularly in women. These findings suggest a possible role for leptin in the control of adipose tissue distribution and mass.

A 5‐hydroxytryptamine receptor in human atrium

1 The effects of 5‐hydroxytryptamine (5‐HT) were investigated on right atrial appendages obtained from patients treated with β‐adrenoceptor blocking agents who were undergoing open heart surgery. Atrial strips were paced under isometric conditions. 2 5‐HT increased contractile force to approximately one half of the force produced by a saturating concentration of (−)−isoprenaline. Both 5‐HT and (−)−isoprenaline accelerated the onset of relaxation, as indicated by an abbreviation of time to peak force. 3 The effects of 5‐HT were resistant to blockade by 0.4 μm (±)‐propranolol, 1μm (−)−pindolol, 0.4 μm methiothepin, 4 μm yohimbine, 0.4 μm ketanserin, 10 μm phenoxybenzamine, 1μm methysergide, 2 μm MDL 72222 and 20 μm granisetron. 4 Cocaine 6 μm potentiated the effects of 5‐HT, increasing the pEC50 from 6.6 to 7.4. The inotropic potency of 5‐HT is five times greater than that of (−)−noradrenaline. 5 ICS 205930 antagonized competitively the effects of 5‐HT with a pKB of 6.7. 6 In the presence of 0.4 μm (±)‐propranolol, 10 μm 5‐HT increased both adenosine 3′:5′ cyclicmonophosphate (cyclic AMP) levels and cyclic AMP‐dependent protein kinase activity by approximately one half and two thirds respectively, of the corresponding effects of 200 μm (−)−isoprenaline. 7 Both the increase in cyclic AMP levels and the stimulation of protein kinase activity are consistent with the inotropic effects of 5‐HT being mediated by cyclic AMP‐dependent phosphorylation of Ca2+ channels and of proteins involved in contraction and relaxation. 8 The human atrial 5‐HT receptor resembles the neuronal ‘so called’ 5‐HT4 receptor of rodents both in increasing cyclic AMP levels and in its affinity for ICS 205930.

Thiazolidinedione exposure increases the expression of uncoupling protein 1 in cultured human preadipocytes

Thiazolidinediones (TZDs) are a novel class of insulinsensitizing agents used in the treatment of NIDDM and are potent agonists for the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ). The thiazolidinedione BRL 49653 has been shown to promote the differentiation of the HIB-1B brown preadipocyte cell line and to increase rat interscapular brown adipose tissue (BAT) mass. Given the importance of brown fat in the control of energy metabolism in rodents, this may represent an important therapeutic effect of this class of compound. To date, however, no studies examining the effects of TZDs on human brown fat have been reported. In the present study, we have measured uncoupling protein 1 (UCP-1) mRNA, a specific marker for BAT, in isolated adipocytes and subcultured preadipocytes prepared from different adult human adipose tissue depots. Consistent with previous studies of adult human whole adipose tissue, UCP-1 mRNA was detectable in isolated human adipocytes prepared from all depots studied with a rank order of perirenal, omental, and subcutaneous. BRL 49653 treatment of subcultured human pre-adipocytes prepared from all depots resulted in increased levels of UCP-1 mRNA, compared with those of the vehicletreatedcells. When exposed to BRL 49653 for 5 days, preadipocytes from the human perirenal depot accumulated lipid, and a proportion of cells showed clear mitochondrial staining for UCP-1 protein by confocal microscopy. Thus, cells of the brown fat lineage were detectable in all human adipose depots studied, and cultured human pre-adipocytes, particularly from the perirenal depot, showed a marked increase in UCP-1 expression in response to thiazolidinediones. Given the role of brown adipocytes in the enhancement of energy expenditure, promotion of brown fat adipogenesis by thiazolidinediones could contribute to the beneficial effects of these drugs on insulin resistance in humans.

Activation of β2-Adrenergic Receptors Hastens Relaxation and Mediates Phosphorylation of Phospholamban, Troponin I, and C-Protein in Ventricular Myocardium From Patients With Terminal Heart Failure

BACKGROUND Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta1- and beta2-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. METHODS AND RESULTS Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 micromol/L) or zinterol (10 micromol/L), mediated through beta2-adrenergic receptors, and of norepinephrine (10 micromol/L), mediated through beta1-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17. Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3; troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3+/-1.4, and 7.5+/-2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta1- and beta2-adrenergic receptors. CONCLUSIONS Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta1- and beta2-adrenergic receptors, thereby potentially improving diastolic function.

A 5-HT4-like receptor in human right atrium

SummaryThe effects of 5-carboxamidotryptamine (5-CT) and the gastrokinetic benzamides renzapride and cisapride on contractile force were investigated using isolated paced right atrial appendages from patients treated with β-adrenoceptor blocking agents who were undergoing open heart surgery. These effects were compared to those of 5-hydroxytryptamine (5-HT). The effects of the drugs on atrial cyclic AMP levels and cyclic AMP-dependent protein kinase ratios were also investigated.The drugs all increased contractile force rank order of potency was 5-HT > renzapride > cisapride > 5-CT. The maximum responses, expressed as a fraction of the response to 200 μmol/l (−)-isoprenaline, were 5-HT 0.6, 5-CT 0.6, renzapride 0.4 and cisapride ≥ 0.2, suggesting that the latter two are partial agonists. 5-HT, 5-CT and renzapride but not cisapride caused significant shortening of time to peak force. The effects of the four drugs were blocked by molar concentrations of ICS 205-930, suggesting an involvement of 5-HT4 receptors. As expected of partial agonists both renzapride and cisapride caused simple competitive antagonism of the positive inotropic effects of 5-HT The estimated equilibrium dissociation constants pKp(−log mol/l Kp) were 6.7 for renzapride and 6.2 for cisapride. 5-CT at concentrations up to 10 μmol/l did not antagonise the effects of 5-HT. In the presence of (±)-propranolol 0.4 μmol/I, 5-HT 10 μmol/l, 5-CT 100 μmol/I, renzapride 10 μmol/l and cisapride 40 μmol/I significantly increased cyclic AMP levels. 5-HT and renzapride also significantly increased cyclic AMP-dependent protein kinase activity, whereas 5-CT caused only marginal stimulation and cisapride was ineffective.The results confirm the existence of a human right atrial 5-HT receptor that is similar in nature to, but not necessarily identical with, the 5-HT4 receptor of mouse embryonic colliculi neurones. The main difference is that in human right atrium the benzamides are less potent and efficacious than 5-HT and that cisapride is less potent and less efficacious than renzapride while in mouse embryonic colliculi these two benzamides are equipotent with and more efficacious agonists than 5-HT We designate the human right atrial 5-HT receptor 5-HT4-like. The human right atrial 5-HT4-like receptor greatly resembles porcine sinoatrial and left atrial 5-HT4-like receptors and also appears to be similar to 5-HT4-like receptors of guinea-pig ileum and rat oesophagus.

5-Hydroxytryptamine causes rate-dependent arrhythmias through 5-HT4 receptors in human atrium: facilitation by chronic β-adrenoceptor blockade

We have investigated the ability of 5-hydroxytryptamine (5-HT) to elicit arrhythmic contractions in isolated human atrial strips as a function of pacing rate (0.1–2 Hz) using a method recently introduced by us (Kaumann and Sanders, this journal, 1993b) and examined the nature of the 5-HT receptors involved. Right atrial appendage tissue was obtained from 14 patients undergoing cardiac surgery. None of the patients had advanced heart failure. 5-HT (0.6–20 μmol/l) induced arrhythmic contractions during pacing in 4/11 atrial strips from 3/4 patients who had not received β blockers and in 21/27 atrial strips from 9/10 patients who had been chronically treated with β blockers (primarily β1-selective). The incidence of arrhythmic contractions evoked by 5-HT did not reach statistical significance in the atrial tissue from the non-β blocked patients but was highly significant in the atrial tissue from the chronically β blocked patients. The arrhythmic contractions usually occurred more frequently at low than at high pacing rates and were observed at the physiological frequency of 1 Hz in 1/4 atrial strips from 1/4 of the non-β blocked patients and 6/11 strips from 5/10 of the β blocked patients. The 5-HT-evoked arrhythmic contractions were observed during blockade of β1-adrenoceptors, β2-adrenoceptors and 5-HT3 receptors, ruling out the participation of these receptors. The 5-HT-evoked arrhythmic contractions were totally inhibited within 30 min by the selective 5-HT4 receptor antagonist SB 203186 ((1-piperidinyl)ethyl 1H-indole 3-carboxylate) 100 nmol/l whereas they persisted in time-matched controls. The blockade of 5-HT-evoked arrhythmic contractions by SB 203186 was surmounted by high concentrations (400–1800 μmol/l) of 5-HT. Our results demonstrate that 5-HT elicits rate-dependent arrhythmic contractions in isolated human atrium through the 5-HT4 receptor and that they are facilitated in atrial tissue from patients treated with β blockers. Our results suggest that endogenous, platelet-derived 5-HT may cause atrial arrhythmias and that exogenous 5-HT4 agonists/partial agonists may be arrhythmogenic.

A 5-HT4-like receptor in human left atrium.

SummaryThe effects of 5-hydroxytryptamine (5-HT) on left atrial preparations obtained from 5 patients with terminal heart failure who were undergoing heart transplant surgery were investigated. The preparations were paced under isometric conditions. In the presence of (−)-pindolol I pmol/l (to block β-adrenoceptors) and cocaine 6 gmol/l (to block tissue uptake of 5-HT) 5-HT increased contractile force with a pEC50 of 7.0. The maximum effect of 5-HT amounted to 24.5% of that caused by a maximally effective concentration of (−)-isoprenaline (200 μmol/l) and 25% of that caused by 6.75 mmol/l CaCl2. The, effects of 5-HT were competitively antagonised by 3α-tropanyl-1H-indole-3-carboxylate (ICS 205–930) with a pKB of 6.8. The effects of 5-HT on cyclic AMP levels and cyclic AMP-dependent protein kinase activity were also studied using left atrial tissues from one of the patients; 5-HT increased the cyclic AMP content and stimulated the kinase. The results are consistent with the existence of a human left atrial 5-HT receptor which is similar to the recently identified human right atrial 5-HT receptor that resembles the 5-HT4 receptor. The left atrial 5-HT4-like receptor is functional in tissues obtained from patients with terminal heart failure.

Both β1- and β2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

SummaryThe involvement of β1- and β2-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with β blockers (usually β1-selective) and 9 patients not treated with β blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. β1-adrenoceptors were activated by (−)-noradrenaline during β2-adrenoceptor blockade with 50 nmol/l ICI 118551. β2-adrenoceptors were activated by (−)-adrenaline during β1-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (−)noradrenaline and (−)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (−)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a β blocker and 17/17 atrial strips from 15/15 patients chronically treated with β blockers. ICI 118551 (50 nmol/l) blocked the (−)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with β blockers and 17/20 atrial strips from 15/18 patients chronically treated with β blockers. The incidence of arrhythmic contractions evoked by both (−)-noradrenaline and (−)-adrenaline was higher in chronically β blocked patients than in non β blocked patients. We conclude that both β1- and β2-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic β1-adrenoceptor blockade.

Sensitization of Human Atrial 5-HT4 Receptors by Chronic β-Blocker Treatment

Background Chronic treatment of patients with β-blockers induces β2-adrenergic receptor hyperresponsiveness in atrium and sinoatrial node. To investigate whether other atrial Gs protein–coupled receptors also become hyperresponsive after chronic treatment with β-blockers, we investigated 5-HT4 receptors in tissues and myocytes, which mediate serotonin-evoked increases of both contractile force and cAMP levels. Methods and Results Isolated right atrial strips from patients who had been chronically treated or not treated with a β-blocker were set up to contract. In tissues from β-blocker–treated patients (n=27), the maximum inotropic response to serotonin was 56±3% (mean±SEM) of the effect elicited by (−)-isoproterenol (200 μmol/L) compared with a response of 19±6% in tissues from non–β-blocker–treated patients (n=13) (P<.001). The responsiveness of the tissues to Ca2+ was unchanged by chronic β-blocker treatment. Serotonin (1 and 10 μmol/L) increased tissue cAMP levels, the increase with 10 μmol/L being si...