1 TNO Prevention and Health, Gaubius Laboratory, Leiden, the Netherlands
2 Department of Internal Medicine, Leiden University Medical Center, Leiden, the Netherlands
3 Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, the Netherlands
4 Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands
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ABSTRACT |
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Peptide YY (PYY) belongs to a family of peptides that is critically involved in the regulation of appetite. It is synthesized in specialized cells (L-cells) that are found primarily in the distal gastrointestinal tract. Circulating PYY levels rise within 15 min after a meal in proportion to the amount of calories ingested and remain elevated for 6 h (1). The two natural forms of this peptide, PYY136 and PYY336, have opposing effects on food intake (2): PYY136 stimulates appetite, whereas PYY336 (the major circulating form) inhibits feeding (35).
PYY336 reduces food intake by acting on appetite regulation centers in the hypothalamus (3,6). Specifically, PYY336 is an agonist of the presynaptic neuropeptide Y (NPY) Y2 receptor, which inhibits NPY neuronal activity in the arcuate nucleus and thereby activates adjacent pro-opiomelanocortin (POMC) neurons (3). In addition to their critical role in the control of feeding, both NPY and POMC affect insulin action. Intracerebroventricular infusion of NPY induces hyperinsulinemia and insulin resistance in rats (7,8). In contrast, central administration of -melanocyte-stimulating hormone, a splice product of the POMC peptide, enhances insulin sensitivity (9). In view of the fact that PYY336 inhibits NPY neuronal activity and activates that of POMC, we wondered whether it could improve insulin sensitivity "directly" (i.e., through a mechanism independent of food intake and body weight). To address this question, we infused PYY336 intravenously and quantified its acute effects on glucose and fatty acid flux during a hyperinsulinemic-euglycemic clamp in mice without access to food during the experiment.
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RESEARCH DESIGN AND METHODS |
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Hyperinsulinemic-euglycemic clamp.
A total of 36 mice were fasted overnight with food withdrawn at 5:00 P.M. the day before the study. The next day, hyperinsulinemic-euglycemic clamps were performed as described earlier (10). PYY or vehicle was administered as a primed (0.15 µg)-continuous (0.25 µg/h) intravenous infusion during the whole experiment (basal and hyperinsulinemic period). In one series of experiments, glucose turnover was quantified, and in another series, free fatty acid (FFA) turnover was determined. First, basal rates of glucose or FFA turnover were measured by giving a primed-continuous infusion of 3H-glucose (prime: 0.7 µCi; continuous: 1.2 µCi/h; Amersham, Little Chalfont, U.K.) or 14C-palmitate (prime: 1.8 µCi; continuous: 3 µCi/h; Amersham), respectively, for 80 min. Subsequently, insulin was administered in a primed (4.1 mU)-continuous (6.8 mU/h) intravenous infusion for 23 h to attain steady-state circulating insulin levels of 6 ng/ml. A variable infusion of 12.5% D-glucose was used to maintain euglycemia, measured at 10-min intervals via tail bleeding (Freestyle; TheraSense, Disetronic Medical Systems, Vianen, the Netherlands). Blood samples (75 µl) were taken during the basal period (after 60 and 80 min) and during the clamp period (when glucose levels were stable and 20 and 40 min later) for determination of plasma glucose, FFA, and insulin concentrations and [3H]glucose and [14C]palmitate specific activities.
To assess insulin-mediated glucose uptake in individual tissues, 2-deoxy-D-[3H]glucose (2-DG; Amersham) was administered as a bolus (1 µCi) 40 min before the end of the clamp experiments in which FFA turnover was measured. At the end of the clamp, mice were killed, and muscle and adipose tissue were isolated and frozen in liquid nitrogen for subsequent analysis.
Analytical procedures.
Plasma levels of glucose and FFAs were determined using commercially available kits (Instruchemie, Delfzijl, the Netherlands, and Wako, Neuss, Germany). Plasma insulin and PYY336 concentrations were measured by a mouse insulin enzyme-linked immunosorbent assay and PYY336 radioimmunoassay (Alpco, Windham, NH, and Phoenix Pharmaceuticals, Belmont, CA). Total plasma 3H-glucose was determined in 7.5 µl plasma and in supernatants after trichloroacetic acid (20%) precipitation and water evaporation to eliminate tritiated water. Total plasma 14C-palmitate was determined in 7.5 µl plasma after extraction of lipids by a modification of Bligh and Dyers (11) method. Briefly, 7.5 µl plasma was dried and resolved in 100 µl water. Then, 1.1 ml demi-water and 4.5 ml methanol:chloroform (2:1) were added and mixed thoroughly, after which 1.5 ml chloroform was added and mixed, and, finally, 1.5 ml demi-water was added and mixed. After centrifugation, the chloroform layer was collected, and the FFA fraction was separated from other lipid components by thin-layer chromatography on silica gel plates.
Tissue analysis.
For determination of tissue 2-DG uptake, the homogenate of muscle and adipose tissue was boiled, and the supernatant was subjected to an ion-exchange column to separate 2-DG-6-phosphate from 2-DG as described previously (10,12,13).
Calculations.
Turnover rates of glucose and FFAs (µmol · min1 · kg1) were calculated during the basal period and in steady-state clamp conditions as the rate of tracer infusion (dpm/min) divided by the plasma specific activity of 3H-glucose or 14C-palmitate (dpm/µmol). The ratio was corrected for body weight. Endogenous glucose production (EGP) was calculated as the difference between the tracer-derived rate of glucose appearance and the glucose infusion rate.
Tissue-specific glucose uptake in muscle and adiopose tissue was calculated from tissue 2-DG content, corrected for plasma specific activity and expressed as micromoles per gram of tissue.
Statistical analysis.
Differences between groups were determined by Mann-Whitneys nonparametric test for two independent samples. P < 0.05 was considered statistically significant. All values shown represent means ± SD.
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RESULTS |
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Plasma parameters.
Plasma glucose FFA, insulin, and PYY336 concentrations in basal and hyperinsulinemic conditions are shown in Table 1. In the basal state, plasma parameters did not differ between PYY- and vehicle-infused animals, except for the plasma PYY336 concentration. Under steady-state clamp conditions, plasma insulin and glucose concentrations were similar in both groups. Hyperinsulinemia suppressed FFA levels to a similar extent in PYY- and vehicle-infused mice.
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DISCUSSION |
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PYY336 clearly enhanced insulin-induced glucose disposal as determined by tracer dilution methodology. Accordingly, 2-DG uptake in muscle and adipose tissue under hyperinsulinemic conditions was higher during PYY336 infusion, albeit the difference with control attained statistical significance only for muscle. In contrast, PYY336 did not significantly impact the capacity of insulin to inhibit EGP. Insulin action on FFA metabolism was not affected by PYY336, either, as indicated by similar fatty acid turnover rates during hyperinsulinemia in PYY336- and saline-infused animals. In light of the current experimental context, these data suggest that PYY336 reinforces the impact of insulin on glucose disposal through a mechanism that is independent of food intake and body weight. In contrast, it appears to leave insulin action on glucose production and FFA turnover largely unaffected.
The plasma PYY336 concentration rose to 3.2 ± 0.7 pg/µl in response to PYY infusion. Relatively few studies have looked at the physiology of circulating PYY336 in rodents. According to a recent article by Batterham et al. (3), postprandial PYY336 concentrations amount to 112 pmol/l (0.4 pg/µl) in freely feeding rats. In a study by Lee et al. (14), plasma PYY levels in fasting mice were 0.18 pg/ml, which accords with our data (Table 1). We are not aware of any study measuring postprandial PYY336 concentrations in mice. Thus, the dose of PYY336 we administered may have induced supraphysiological PYY336 levels. Therefore, our data do not allow a definitive inference as to whether the postprandial rise of circulating PYY336 can reinforce insulin action.
Postprandial PYY336 release is decreased in obese compared with lean subjects, and circulating levels correlate negatively with BMI. Conversely, intravenous PYY336 infusion significantly reduces food intake in humans (15), and repeated administration of PYY336 attenuates weight gain in rodents (3). These findings suggest that diminished PYY336 release may contribute to the pathogenesis of obesity in animals and humans. The observations presented here allow us to hypothesize that low circulating PYY336 levels may also compromise insulin action in obese subjects. Moreover, perhaps even more important, the data suggest that PYY336 or synthetic analogs of this peptide may be useful tools in the clinical management of obesity and insulin resistance.
It remains to be established whether PYY336 acts through hypothalamic neural circuits (by analogy with the mechanism guiding its effects on appetite) to enhance insulin-mediated glucose disposal. Because PYY336 is a high-affinity agonist to the Y2 receptor (2) and the distribution of this receptor subtype is largely confined to the central nervous system (particularly the arcuate nucleus of the hypothalamus) (16), it is most likely that PYY336 modulates insulin action by activation of Y2 receptors in the brain. As alluded to earlier, Y2 receptor signaling inhibits NPY neuronal activity in the arcuate nucleus of the hypothalamus and thereby activates adjacent POMC neurons (3). Hypothalamic overexpression of NPY induces obesity and insulin resistance in mice (7,8), whereas activation of melanocortin receptor subtypes 3 and 4 in the brain enhances insulin sensitivity (9). Thus, the present data are in keeping with the contention that PYY336 modulates insulin action via hypothalamic Y2 receptor. The downstream mechanisms that actuate the effects of hypothalamic neuronal circuits on muscle and adipose tissue remain to be fully elucidated. At this point, vagotomy was shown to prevent the hyperinsulinemic effects of NPY, which suggests that the autonomic nervous system may be involved (17). Also, adrenalectomy prevents the obesity syndrome produced by chronic central NPY infusion and reverses the obese phenotype in leptin-deficient ob/ob mice (18,19), indicating that the pituitary-adrenal ensemble may also serve as a second messenger. Thus, neural and/or endocrine mechanistic routes may convey signals from hypothalamic nuclei to peripheral tissues to orchestrate energy balance and fuel flux. It is conceivable that similar routes partake in the control of insulin action by PYY336.
In conclusion, PYY336 reinforces insulin action in mice maintained on a high-fat diet through a mechanism that is independent of food intake and body weight. In this context, PYY336 appears to predominantly impact insulin-mediated glucose disposal, whereas it leaves insulin action on glucose production largely unaffected. These novel data suggest that PYY336 or synthetic analogs of this peptide may be valuable assets to our armamentarium of drugs designed to battle insulin resistance and type 2 diabetes.
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ACKNOWLEDGMENTS |
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This study is conducted in the framework of the Leiden Center for Cardiovascular Research LUMC-TNO.
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FOOTNOTES |
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Address correspondence and reprint requests to Hanno Pijl, MD, Leiden University Medical Center, Department of Endocrinology and Metabolic Diseases, P.O. Box 9600, 2300 RC Leiden, Netherlands. E-mail: h.pijl{at}lumc.nl
Received for publication March 31, 2004 and accepted in revised form May 12, 2004
2-DG, 2-deoxy-D-[3H]glucose; EGP, endogenous glucose production; FFA, free fatty acid; NPY, neuropeptide Y; POMC, proopiomelanocortin; PYY, peptide YY
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REFERENCES |
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