Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of diet-induced obesity.

AIMS/HYPOTHESES: High-fat diets produce obesity and glucose intolerance by promoting the development of insulin resistance in peripheral tissues and liver. The present studies sought to identify the initial site(s) where insulin resistance develops using a moderately high-fat diet and to assess whether the bioflavonoid, quercetin, ameliorates progression of this sequence. METHODS: Four cohorts of male C57BL/6J mice were placed on diets formulated to be low-fat (10% of energy from fat), high-fat (45% of energy from fat) or high-fat plus 1.2% quercetin (wt/wt). After 3 and 8 weeks, cohorts were evaluated using euglycaemic-hyperinsulinaemic clamps, metabolomic analysis of fatty acylcarnitines and acute in vitro assessments of insulin signalling among tissues. RESULTS: After 3 and 8 weeks, the high-fat diet produced whole-body insulin resistance without altering insulin-dependent glucose uptake in peripheral tissues. The primary defect was impaired suppression of hepatic glucose production by insulin at both times. Quercetin initially exacerbated the effect of high-fat diet by further increasing hepatic insulin resistance, but by 8 weeks insulin resistance and hepatic responsiveness to insulin were similarly compromised in both high-fat groups. The high-fat diet, irrespective of quercetin, increased short-chain fatty acylcarnitines in liver but not in muscle, while reciprocally reducing hepatic long-chain fatty acylcarnitines and increasing them in muscle. CONCLUSIONS/INTERPRETATION: Failure of insulin to suppress hepatic glucose output is the initial defect that accounts for the insulin resistance that develops after short-term consumption of a high-fat (45% of energy) diet. Hepatic insulin resistance is associated with accumulation of short- and medium-, but not long-chain fatty acylcarnitines. Dietary quercetin does not ameliorate the progression of this sequence.

The expression and function of G-proteins in experimental intimal hyperplasia.

G-proteins are membrane-bound signal transduction proteins which couple extracellular receptor signals to various effectors. This study examines the expression and the function of G-proteins (alpha i, alpha s, alpha q, and alpha o) in experimental intimal hyperplasia. Vein bypass grafts were placed in 30 New Zealand White rabbits and were harvested after 28 d. The contralateral jugular veins served as controls. Isometric tension studies were performed on rings from veins and vein grafts (n = 10), and Western blot and mRNA analyses were performed in another 20 vessels. There was a fivefold increase in alpha q, a 2.7-fold increase in the alpha i2, and a 3.3-fold increase in alpha s expressions in vein grafts compared with veins. Detectable expression of alpha i3 was observed in vein grafts but not in jugular veins. In addition, there was a 3.8-fold increase in beta subunits in the vein grafts compared with the veins. mRNA for alpha s, alpha i3, and alpha i2 were all elevated in the vein grafts. No detectable levels of the alpha i1 protein or its mRNA were present in either veins or vein grafts. Contractile responses in the veins were not inhibited by pertussis toxin. The contractile responses to norepinephrine were enhanced by twofold, and the responses to serotonin developed de novo in vein grafts compared with veins. The contractile responses to both norepinephrine and serotonin were only partially inhibited by pertussis toxin in the vein grafts even though there was 100% ADP ribosylation with pertussis toxin in both veins and vein grafts. These data suggest that intimal hyperplasia is associated with increased or novel expression of G-proteins in vivo which occur simultaneously with the development of pertussis toxin-sensitive contractile responses. Changes in G-proteins at a transcriptional level or at the level of RNA stability may be involved in the response of smooth muscle cells to injury and to intimal hyperplasia formation.

Alterations in macrophage G proteins are associated with endotoxin tolerance.

Previous studies have suggested that endotoxin tolerance induces macrophage desensitization to endotoxin through altered guanine nucleotide regulatory (G) protein function. In the present study the binding characteristics of the nonhydrolyzable GTP analogue GTP gamma [35S] to macrophage membranes from endotoxin tolerant and control rats were determined. Membranes were prepared from peritoneal macrophages harvested from rats 72 h after two sequential daily doses of vehicle or Salmonella enteritidis endotoxin (100 micrograms/kg on day 1 and 500 micrograms/kg on day 2). GTP gamma [35S] bound to a single class of sites that were saturable and displaceable in control and endotoxin tolerant macrophage membranes. The maximum specific binding of GTP gamma [35S] was significantly (P < 0.01) decreased in membranes from tolerant rats compared to control (Bmax = 39 +/- 7 pmol/mg protein in control vs. 11 +/- 2 pmol/mg protein in endotoxin tolerant; n = 5). There were no significant differences in the Kd values. To determine whether the reduced GTP gamma S binding was due to decreases in G proteins, macrophage membrane G protein content was determined by western blotting with specific antisera to Gi1,2 alpha, Gi3 alpha, Gs alpha, and the beta subunit of G. Scanning densitometric analysis demonstrated differential decreases in tolerant macrophage membrane G proteins. Gi3 alpha was reduced the most to 48 +/- 8% of controls (n = 3), and this reduction was significant compared to those of other G proteins. Gi1,2 alpha and G beta were reduced to 73 +/- 5% (n = 3) and 65 +/- 4% (n = 3) of control values, respectively. Gs alpha(L) and Gs alpha(H) were reduced to 61 +/- 5% (n = 3) and 68 +/- 3% (n = 3) of control, respectively. These results demonstrate that endotoxin tolerant macrophages exhibit decreased membrane GTP binding capacity and differential reductions in the content of specific G proteins. The cellular mechanisms leading to such alterations in G proteins and their functional significance in the acquisition of endotoxin tolerance merit further investigation.

Somatostatin selectively couples to G(o) alpha in HIT-T15 cells.

Previously, we have demonstrated that somatostatin mediates all of its inhibitory effects on glucose-induced insulin secretion from the HIT-T15 cell through pertussis toxin-sensitive G-proteins and that the membrane fraction of this clonal line of pancreatic beta-cells contains six such proteins: G(i) alpha 1, G(i) alpha 2, G(i) alpha 3, and three forms of G(o) alpha. To determine the specificity of somatostatin receptor-G-protein coupling in HIT-T15 cells, we examined the ability of antisera specific for the COOH-terminus of G alpha subtypes to inhibit somatostatin-induced augmentation of membrane GTPase activity. GTPase activity increased in membranes as a function of GTP. At all concentrations of GTP studied, 1 mumol/l somatostatin stimulated GTPase activity. Pertussis-toxin pretreatment prevented the effects of somatostatin. Antisera selective for G(o) alpha subtypes reduced the effects of somatostatin on GTPase activity (GTPase activity in absence of antisera, 125 +/- 3% of control; in the presence of antisera 976, 110 +/- 2% of control; n = 13, P < 0.001), whereas antisera directed against G(i) alpha 1, G(i) alpha 2, G(i) alpha 3, and Gs alpha were without effect. Somatostatin also significantly prevented cyclic AMP accumulation during perifusion with 11.1 mmol/l glucose through a pertussis toxin-sensitive mechanism. These data indicate that the somatostatin receptor couples to G(o) alpha in the HIT-T15 cell and suggest that G(o) alpha may link somatostatin to cyclic AMP metabolism in pancreatic beta-cells.

Multiplicity of mechanisms of serotonin receptor signal transduction.

The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.

Impaired expression and functional activity of the beta 3- and beta 1-adrenergic receptors in adipose tissue of congenitally obese (C57BL/6J ob/ob) mice.

Adipocytes from genetically obese (ob/ob) mice display an impaired response to beta-adrenergic stimulation, but the molecular defects have not been unequivocally identified. The expression and functional activity of the beta 1-, beta 2-, and beta 3-adrenergic receptor (AR) subtypes in white and brown adipose tissue from genetically lean and obese (ob/ob) mice were compared. Three beta 3AR transcripts of 2.1, 2.6, and 3.5 kilobases were identified in adipose tissue from lean mice by Northern blotting. All three beta 3AR mRNA species were dramatically reduced (by approximately 300-fold) in 12-week-old obese mice compared to those in lean animals. beta 1AR mRNA levels were also reduced (by approximately 4-fold) in obese mice, whereas beta 2AR mRNA levels were not significantly changed. The functional consequences of these changes in beta 3AR and beta 1AR expression were assessed by measuring beta-agonist-stimulated adenylyl cyclase activity in adipocyte plasma membranes with subtype-selective beta-adrenergic agonists and antagonists. Dose-response curves with epinephrine from lean mice were best fit to a two-component model comprised of 23% high affinity (K(act) = 1.42 x 10(-7) M) and 77% low affinity (K(act) = 1.67 x 10(-5) M) components, corresponding to activation of beta 1AR and beta 2AR conjointly, and beta 3AR, respectively. The beta 1AR-selective antagonist CGP20712A reduced the high affinity component to about 10%, whereas the nonselective beta-antagonist propranolol eliminated the high affinity component. The beta 3AR-selective agonist BRL37344 stimulated adenylyl cyclase activity in lean membranes to a slightly lesser extent than epinephrine, but was more potent (73% high affinity component; K(act) = 3.61 x 10(-8) M). In obese mice, stimulation of adenylyl cyclase by all agonists was severely blunted and was best fit to a single class of sites. Studies with CGP20712A or the beta 2AR-selective antagonist ICI118,551 indicated that this residual response was predominantly beta 2AR in character. Expression of beta AR subtypes in both brown and white adipose tissue of weanling obese mice (4-5-weeks of age) was also affected, but to a lesser extent, consistent with the progressive severity of obesity with age. Together the reduction in expression of the beta 3AR and beta 1AR impairs the beta-agonist-stimulated adenylyl cyclase response over a broad concentration range by greatly lowering the maximum stimulation and shifting the adrenergic sensitivity at low concentrations from a mixed beta 1AR/beta 2AR response to predominantly beta 2AR.

In vivo effects of dietary quercetin and quercetin-rich red onion extract on skeletal muscle mitochondria, metabolism, and insulin sensitivity.

Red onions and low doses of the flavonoid, quercetin, increase insulin sensitivity and improve glucose tolerance. We hypothesized that dietary supplementation with red onion extract (RO) would attenuate high fat diet (HFD)-induced obesity and insulin resistance similar to quercetin supplementation by increasing energy expenditure through a mechanism involving skeletal muscle mitochondrial adaptations. To test this hypothesis, C57BL/6J mice were randomized into four groups and fed either a low fat diet (LF), HFD (HF), HFD + quercetin (HF + Q), or HFD + RO (HF + RO) for 9 weeks. Food consumption and body weight and composition were measured weekly. Insulin sensitivity was assessed by insulin and glucose tolerance tests. Energy expenditure and physical activity were measured by indirect calorimetry. Skeletal muscle incomplete beta oxidation, mitochondrial number, and mtDNA-encoded gene expression were measured. Quercetin and RO supplementation decreased HFD-induced fat mass accumulation and insulin resistance (measured by insulin tolerance test) and increased energy expenditure; however, only HF + Q showed an increase in physical activity levels. Although quercetin and RO similarly increased skeletal muscle mitochondrial number and decreased incomplete beta oxidation, establishing mitochondrial function similar to that seen in LF, only HF + Q exhibited consistently lower mRNA levels of mtDNA-encoded genes necessary for complexes IV and V compared to LF. Quercetin- and RO-induced improvements in adiposity, insulin resistance, and energy expenditure occur through differential mechanisms, with quercetin-but not RO-induced energy expenditure being related to increases in physical activity. While both treatments improved skeletal muscle mitochondrial number and function, mtDNA-encoded transcript levels suggest that the antiobesogenic, insulin-sensitizing effects of purified quercetin aglycone, and RO may occur through differential mechanisms.

Paradoxical effects of dopamine (DA): Gi alpha 3 mediates DA inhibition of PRL release while masking its PRL-releasing activity.

In an attempt to elucidate the mechanism(s) underlying the paradoxical (inhibitory vs. stimulatory) effects of DA on PRL release, we treated permeabilized pituitary cells with antibodies directed against the carboxyl-terminus of the alpha-subunit of the guanine nucleotide binding protein, Gi3. Immunoneutralization of Gi alpha 3 completely blocked the inhibitory effect of 1000 nM DA on PRL release, as assessed by reverse hemolytic plaque assays. In contrast, DA at a 100-fold lower concentration (10 nM) had no effect on PRL release under control conditions, but elicited a stimulatory response in the presence of anti-Gi alpha 3. Examination of the frequency distribution of plaque sizes indicated that suppression and augmentation of PRL secretion by DA was not attributable to distinct subpopulations of lactotropes. Taken together, these data suggest that all pituitary lactotropes have the potential to respond to the inhibitory and stimulatory activities of DA. However, the stimulatory action of this monoamine is normally masked by tonic activation of Gi alpha 3 which couples DA to its inhibitory pathway.

The beta 3-adrenergic receptor inhibits insulin-stimulated leptin secretion from isolated rat adipocytes.

Various model systems have been used to study the expression of the recently cloned ob gene, leptin. Here we report that freshly isolated rat white adipocytes incubated with insulin release leptin in a rapid and concentration-dependent manner (EC50 of 0.221 +/- .075 nM). Insulin-stimulated leptin release could be detected as early as 30 min and a maximal 2-3 fold effect was produced by 10 nM insulin. The effect of insulin was completely blocked by simultaneous activation of cAMP-dependent protein kinase. Using the activation of lipolysis as an index of cAMP-dependent protein kinase activity, we show that inhibition of leptin release by norepinephrine or the selective beta 3-adrenergic receptor agonist, CL316,243, occurred in parallel to activation of cAMP-dependent protein kinase. In addition, beta 1- and beta 2-adrenergic receptor antagonists did not impair the ability of norepinephrine or CL316,243 to inhibit leptin release from the adipocytes. These findings suggest that the beta 3-adrenergic receptor plays a central role in regulating the release of leptin from the adipocyte.

Adrenalectomy after weaning restores beta3-adrenergic receptor expression in white adipocytes from C57BL/6J-ob/ob mice.

The role of hypercorticism in the development of compromised beta-adrenergic signaling in adipose tissue was assessed in ob/ob mice adrenalectomized at 4 weeks of age and studied 1 and 3 weeks thereafter. Adrenalectomy prevented the rapid increase in body weight and fat deposition between 4 and 5 weeks of age in ob/ob mice and produced a phenotype indistinguishable from that of lean mice. However, adrenalectomized ob/ob mice became intermediate between lean and ob/ob mice by 7 weeks of age. Adipocyte beta3-adrenergic receptor (AR) messenger RNA levels were similar between lean and adrenalectomized ob/ob mice at both time points and were 4- to 8-fold higher than messenger RNA levels in ob/ob mice. As judged by maximal activation of adenylyl cyclase by a beta3-AR-selective agonist, adrenalectomy also restored functional activity of the beta3-AR to levels above or equivalent to those seen in lean mice at both time points. The present results suggest that development of hypercorticism at or before weaning in ob/ob mice represses expression of the beta3-AR and prevents the normal postweaning development of this signaling system in the adipocyte.

Peptide-YY, a new partner in the negative feedback control of pancreatic secretion.

Peptide YY (PYY), a newly discovered ileocolonic peptide, is released by nutrients in the proximal and distal intestine and inhibits pancreatic secretion. However, it is not clear whether PYY can be released in the absence of nutrients in the intestine or whether a physiological role exists for endogenous PYY in negative feedback regulation of pancreatic secretion by pancreatic proteases. In the present study we measured plasma PYY concentrations and determined the effects of anti-PYY serum during stimulation of pancreatic secretion by pancreatic juice diversion (PJD). The effect of SMS 201-995 (SMS; an analog of somatostatin), another inhibitor of pancreatic secretion, on regulation of PYY release induced by PJD was also investigated. Male Wistar rats equipped with pancreatic, biliary, duodenal, and jugular venous cannulas were studied 4-6 days postoperatively. After 90 min of basal collection, pancreatic juice was diverted for 4 h with or without infusion of SMS (2 micrograms/kg.h), given either iv or intraduodenally (ID). Plasma PYY concentrations were significantly increased from a basal level of 177 +/- 15 pg/ml to a peak level of 328 +/- 43 pg/ml 2 h after PJD. These increases in PYY concentration paralleled those in pancreatic protein and fluid outputs. Both iv and ID infusion of SMS during the first 2 h of PJD markedly decreased the plasma PYY concentration to 134 +/- 27 pg/ml and 156 +/- 19 pg/ml, respectively; the total incremental PYY release during 4 h of PJD was inhibited by 100% and 84% by iv and ID SMS, respectively. One milliliter of anti-PYY serum given iv significantly augmented the increment in protein and fluid output during PJD. These results suggest that endogenous PYY released by PJD may play a physiological role in negative feedback regulation of pancreatic secretion in rats.

Age-dependent changes in beta-adrenergic receptor subtypes and adenylyl cyclase activation in adipocytes from Fischer 344 rats.

Epididymal adipocytes were isolated from Fischer 344 rats aged 3, 6, 12, and 24 months, to study the mechanisms responsible for age-dependent diminution in cellular adrenergic responsiveness. Messenger RNA (mRNA) levels for the beta 1-, beta 2-, and beta 3-adrenergic receptors (ARs) were compared across age groups and related to adenylyl cyclase activation by selective receptor agonists in adipocyte plasma membranes and activation of lipolysis in intact cells. mRNA levels for the beta 1-AR decreased by 60% between 3-6 months and remained at this reduced level through 12 and 24 months. A modest increase in beta 2-AR mRNA was noted between 3-12 months, but decreased between 12-24 months to levels seen in the 3-month-old group. mRNA for the beta 3-AR did not change between 3-6 months, but decreased by about 40% between 6-12 months, and by a further 50% between 12-24 months. Lipolytic responsiveness also diminished with age, and regardless of whether beta 3-selective or beta 1/beta 2-selective agonists were used, the maximal release of glycerol was most severely blunted in adipocytes from 24-month-old rats. The age-dependent changes in adenylyl cyclase activation by beta-adrenergic agonists mirrored the observed changes in lipolytic responsiveness with respect to diminished efficacy. These results together with the similar forskolin-stimulated adenylyl cyclase activity among the groups suggest age-dependent changes in activation of adenylyl cyclase at a prior step. This suggestion is also supported by the comparable inhibitory capacities of the alpha 2-adrenergic and A1-adenosine signaling systems among the age groups. In view of the similar levels of Gs alpha, the age-dependent decrease in adrenergic responsiveness in rat adipocytes appears to result primarily from specific decreases in the expression of both beta 3-AR and beta 1-ARs.

Induction of uncoupling protein expression in brown and white adipose tissue by leptin.

Deposition of excess body fat occurs when energy intake chronically exceeds energy expenditure. In ob/ob mice, the absence of leptin affects both components of the energy balance equation, and the mice become morbidly obese after weaning. Treatment of ob/ob mice with exogenous leptin reduces body weight by decreasing food intake and stimulating energy utilization, but even when saline- and leptin-injected ob/ob mice are pair-fed, mice receiving leptin lose significantly more weight. Therefore, the purpose of the present study was to test the hypotheses that uncoupling protein-1 (UCP1) expression is reduced in adipose tissue from ob/ob mice and is restored by treatment with exogenous leptin. Lean and ob/ob mice (5-6 weeks old) were housed at 23 C and treated with leptin (20 microg/g BW x day) for 3 days before they were killed. Compared with levels in lean littermates, UCP1 messenger RNA (mRNA) and protein levels were lower in brown adipose tissue (BAT) and retroperitoneal white adipose tissue (WAT) from ob/ob mice. Treatment of ob/ob mice with leptin reduced body weight and produced a 4- to 5-fold increase in UCP1 mRNA levels in both interscapular BAT and retroperitoneal WAT. The increases in UCP1 mRNA were accompanied by comparable increases in UCP1 protein in mitochondrial preparations from each tissue. Given that the sole known function of UCP1 is to uncouple oxidative phosphorylation, the present results are consistent with the conclusion that leptin stimulates energy utilization in ob/ob mice by increasing thermogenic activity and capacity (UCP1). In addition, the present results suggest that decreased UCP1 expression in BAT and WAT of ob/ob mice is in part responsible for their increased metabolic efficiency and propensity to become obese.

Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue.

Exogenous leptin enhances energy utilization in ob/ob mice by binding its hypothalamic receptor and selectively increasing peripheral fat oxidation. Leptin also increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), but the neurotransmitter that mediates this effect has not been established. The present experiments sought to determine whether leptin regulates UCP1 expression in BAT and its own expression in white adipose tissue (WAT) through the long or short forms of leptin receptor and modulation of norepinephrine release. Mice lacking dopamine beta-hydroxylase (Dbh-/-), the enzyme responsible for synthesizing norepinephrine and epinephrine from dopamine, were treated with leptin (20 microg/g body weight/day) for 3 days before they were euthanized. UCP1 messenger RNA (mRNA) and protein expression were 5-fold higher in BAT from control (Dbh+/-) compared with Dbh-/- mice. Leptin produced a 4-fold increase in UCP1 mRNA levels in Dbh+/- mice but had no effect on UCP1 expression in Dbh-/-. The beta3-adrenergic agonist, CL-316,243 increased UCP1 expression and established that BAT from both groups of mice was capable of responding to beta-adrenergic stimulation. Similarly, exogenous leptin reduced leptin mRNA in WAT from Dbh+/- but not Dbh-/- mice. In separate experiments, leptin produced comparable reductions in food intake in both Dbh+/- and Dbh-/- mice, illustrating that norepinephrine is not required for leptin's effect on food intake. Lastly, db/db mice lacking the long form of the leptin receptor failed to increase UCP1 mRNA in response to exogenous leptin but increased UCP1 mRNA in response to CL-316,243. These studies establish that norepinephrine is required for leptin to regulate its own expression in WAT and UCP1 expression in BAT and indicate that these effects are likely mediated through the centrally expressed long form of the leptin receptor.

The metabolic significance of leptin in humans: gender-based differences in relationship to adiposity, insulin sensitivity, and energy expenditure.

Leptin is an adipocyte-derived hormone that interacts with a putative receptor(s) in the hypothalamus to regulate body weight. The relationship of leptin to metabolic abnormalities associated with obesity together with hormonal and substrate regulation of leptin have not been extensively studied. Therefore, 116 subjects (62 men and 54 women) with a wide range of body weight [body mass index (BMI), 17-54 kg/m2] were characterized on a metabolic ward with regard to body composition, glucose intolerance, insulin sensitivity, energy expenditure, substrate utilization, and blood pressure. Eighty-five of the subjects had normal glucose tolerance (50 men and 35 women), and 31 had noninsulin-dependent diabetes mellitus (12 men and 19 women). In both men and women, fasting leptin levels were highly correlated with BMI (r = 0.87 and r = 0.88, respectively) and percent body fat (r = 0.82 and r = 0.88, respectively; all P < 0.0001). However, men exhibited lower leptin levels at any given measure of obesity. Compared with those in men, leptin levels rose 3.4-fold more rapidly as a function of BMI in women [leptin = 1.815 (BMI)-31.103 in women; leptin = 0.534 (BMI)-8.437 in men] and 3.2 times more rapidly as a function of body fat [leptin = 1.293 (% body fat)-24.817 in women; leptin = 0.402 (% body fat)-3.087 in men]. Hyperleptinemia was associated with insulin resistance (r = -0.57; P < 0.0001) and high waist to hip ratio (r = 0.75; P < 0.0001) only in men. On the other hand, during the hyperinsulinemic euglycemic clamp studies, hyperinsulinemia acutely increased leptin concentrations (20%) only in women. There was no correlation noted between fasting leptin levels and either resting energy expenditure or insulin-induced thermogenesis in men or women (P = NS). In stepwise and multiple regression models with leptin as the dependent variable, noninsulin-dependent diabetes mellitus did not enter the equations at a statistically significant level. The data indicate that there are important gender-based differences in the regulation and action of leptin in humans. Serum leptin levels increase with progressive obesity in both men and women. However, for any given measure of obesity, leptin levels are higher in women than in men, consistent with a state of relative leptin resistance. These findings have important implications regarding differences in body composition in men and women. The observation that serum leptin is not related to energy expenditure rates suggests that leptin regulates body fat predominantly by altering eating behavior rather than calorigenesis.

Regulation of pancreatic endocrine function by cholecystokinin: studies with MK-329, a nonpeptide cholecystokinin receptor antagonist.

A cholecystokinin (CCK) receptor antagonist, MK-329, was used to explore the physiological role of CCK in regulating pancreatic endocrine function in humans. The ability of CCK to increase plasma pancreatic polypeptide (PP) concentrations and blockade of this effect with MK-329 were evaluated in a double blind, balanced, four-period cross-over study. Eight subjects received single oral doses of 0.5, 2, or 10 mg MK-329 or placebo, followed by an iv infusion of CCK-8 (34 ng/kg.h). In placebo-treated subjects, PP increased from basal levels of 70 +/- 15 (+/- SE) to peak values of 291 +/- 58 pg/mL after CCK infusion (P less than 0.05 compared to basal). This increase in plasma PP concentration was inhibited in a dose-dependent fashion by MK-329, with 10 mg antagonizing the stimulatory effect of CCK infusion by nearly 80%. Second, the effect of MK-329 on meal-stimulated pancreatic endocrine responses was evaluated by giving placebo or 10 mg MK-329 2 h before ingestion of a mixed meal. Eight subjects were treated in a randomized two-period cross-over fashion. With placebo treatment, peak postprandial plasma insulin, glucagon, and glucose concentrations were 101 +/- 8 microU/mL, 195 +/- 15 pg/mL, and 150 +/- 10 mg/dL, respectively (all P less than 0.05). The integrated PP response following the meal was 56.3 +/- 11.1 ng/mL.minute. With MK-329 treatment, the integrated PP concentration was reduced to 33.9 +/- 2.2 ng/mL.min (P less than 0.05 compared to placebo treatment). Mean postprandial insulin, glucagon, and glucose concentrations did not differ between placebo and MK-329 treatments. We conclude that CCK receptor blockade with 10 mg MK-329 does not alter plasma insulin, glucagon, or glucose responses to a mixed meal. However, the observation that physiological concentrations of CCK increase plasma levels of PP, and the finding that CCK receptor blockade selectively attenuates the postprandial increase in plasma PP concentrations support a physiological role for CCK in regulating PP secretion.

The recombinant 5-HT1A receptor: G protein coupling and signalling pathways.

The 5-hydroxytryptamine 5-HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5-HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5-HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5-HT1A receptor in the decade since the identification of its cDNA.

Role of guanine nucleotide-binding proteins, Gi alpha 3 and Gs alpha, in dopamine and thyrotropin-releasing hormone signal transduction: evidence for competition and commonality.

It is clear that dopamine (DA) at high concentrations (> 100 nmol/l) inhibits the release of prolactin (PRL). Paradoxically, this monoamine at low concentrations (< 10 nmol/l) has also been shown to augment PRL secretion. One possible explanation for these divergent effects is that DA binds receptors capable of interacting with multiple G protein subtypes that recruit opposing intracellular signaling pathways within lactotropes. To identify G proteins which couple DA receptor activation to PRL secretion, we have selectively immunoneutralized the activity of Gi alpha 3 and Gs alpha in primary cultures of rat pituitaries and subsequently tested the ability of these cultures to respond to high and low dose DA. Specifically, permeabilized pituitary cell cultures from random-cycling female rats were treated with control immunoglobulins (IgGs; 50 micrograms/ml) purified from preimmune serum (PII) or IgGs directed against the C-terminal portion of Gi alpha 3 or Gs alpha. After immunoneutralization of these G proteins, cells were challenged with 10 or 1000 nmol DA/l and the relative amount of PRL released was assessed by reverse hemolytic plaque assay. Results were expressed as % of basal values and compared. Under control conditions (PII), 1000 nmol DA/l inhibited (61.4 +/- 7.6% of basal values; mean +/- S.E.M.) while 10 nmol DA/l augmented (120.0 +/- 7.0%) PRL release in five separate experiments. Treatment of cells with anti-Gi alpha 3 attenuated the inhibitory effect of high dose DA (87.3 +/- 14.5%). However, elimination of Gi alpha 3 activity did not significantly alter the PRL stimulatory effect of 10 nmol DA/l (121.0 +/- 5.2%). Interestingly, immunoneutralization of Gs alpha resulted in a reciprocal shift in the activity of the lower dose of DA from stimulatory to inhibitory (69.7 +/- 7.3%) while combined treatment of anti-Gi alpha 3 and anti-Gs alpha abrogated the responsiveness of pituitary cell cultures to either DA treatment (1000 nmol/l, 70.7 +/- 12.5% and 10 nmol/l, 87.5 +/- 21.4%). These data reveal that ligand-activated DA receptors can interact with both Gi alpha 3 and Gs alpha. Elimination of the stimulatory component (Gs alpha) favors the DA receptor activation of the inhibitory pathway (Gi alpha 3) suggesting a competition between negative and positive intracellular signaling mechanisms in normal lactotropes. In addition to DA treatment, we also challenged permeabilized pituitary cells with 100 nmol thyrotropin-releasing hormone (TRH)/l as a positive control for secretory integrity. As anticipated, TRH stimulated PRL release to 188.0 +/- 31.0% of basal values under control conditions. Unexpectedly, immunoneutralization of Gs alpha completely blocked the ability of TRH to induce PRL release (101.8 +/- 12.0%). This neutralizing effect was specific to Gs alpha in that blockade of Gi alpha 3 activity had no significant effect on TRH-stimulated PRL release (166.2 +/- 13.1%). These data are the first to support a direct role of Gs alpha in TRH signal transduction within PRL-secreting cells.

Suppression of LH secretion by oestradiol, dihydrotestosterone and trenbolone acetate in the acutely castrated bull.

Twenty acutely castrated bulls were used to investigate the role of androgenic and oestrogenic steroids in the feedback control of LH secretion. The effects of 5 alpha-dihydrotestosterone (DHT) or the growth stimulants trenbolone acetate (TBA) or oestradiol-17 beta (OE2) on serum LH secretory profiles were measured. In addition, pituitary LH responses to exogenous LH releasing hormone (LHRH) were determined to differentiate between hypothalamic and pituitary sites of steroid action. At the time of castration, two groups of animals were given implants of either 45 mg OE2 or 200 mg TBA. Another group received equivalent to 30 mg daily injections of DHT. Control steers showed an increase in LH from 2.4 +/- 0.5 (S.E.M.) micrograms/l to 7.0 +/- 0.5 micrograms/l during the week after castration. Treatment with DHT and TBA prevented the post-castration rise in serum LH. In contrast, steers given implants of OE2 showed a significantly greater increase in LH than controls 1 day after castration, but by day 5 LH declined in the OE2-treated group to precastration values. Five weeks after castration control steers secreted LH in pulses at intervals of 40-50 min and with an amplitude of 4.2 +/- 0.4 micrograms/l. Pulses were not detected in the LH profiles of the steroid-treated steers. Dihydrotestosterone and TBA significantly reduced pituitary LH responses to exogenous LHRH, whereas steers receiving OE2 showed LH responses to LHRH which were similar to those observed in castrated controls. These results support the hypothesis that androgenic and oestrogenic components participate separately in the feedback control of LH secretion in the bull.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of secretion by the T84 colonic epithelial cell line with dietary flavonols.

Flavonols are dietary compounds widely distributed in plants and characterized by a 2-phenyl-benzo(alpha)pyrane nucleus possessing hydroxyl and ketone groups at positions 3 and 4, respectively. Kaempferol, quercetin, and myricetin are flavonols that are further mono-, di-, or trihydroxylated on the phenyl ring, respectively. To test whether these ingested flavonols might exert a direct secretory effect on intestinal epithelial cells, monolayers of the T84 colonocyte cell line were mounted in Ussing chambers and examined for ion transport response. Twenty minutes after addition of 100 microM quercetin to either the serosal or mucosal side, the short-circuit current change was maximal at 16.6 microA/cm2. Kaempferol was less potent than quercetin, while myricetin and glycosylated quercetin (rutin) did not induce secretion. The secretion induced by quercetin did not seem to be mediated by the reactive oxygen species generated by quercetin through auto-oxidation and/or redox cycling (superoxide, hydrogen peroxide, and the hydroxyl radical) because it was neither enhanced by iron, nor inhibited by desferroxamine B or catalase (alone or in combination with superoxide dismutase). Like vasoactive intestinal peptide, quercetin induced a secretory response that was inhibited by barium chloride and bumetanide, and which exhibited synergism with carbachol. Quercetin also stimulated a modest increase in intracellular cAMP levels and the phosphorylation of endogenous protein substrates for cAMP-dependent protein kinase. Thus, quercetin is a potent stimulus of colonocyte secretion that resembles secretagogues which act via a cAMP-mediated signaling pathway.