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The effect of different macronutrient infusions on appetite, ghrelin and peptide YY in parenterally fed patients
ARTICLE IN PRESS Clinical Nutrition (2006) 25, 626–633
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
The effect of different macronutrient infusions on appetite, ghrelin and peptide YY in parenterally fed patients Charles D. Murraya,b, Carel W. le Rouxb,c, Catherine Gouveiab, Paul Bassetta, Mohammad A. Ghateib,c, Stephen R. Bloomb,c, Anton V. Emmanuela,b, Simon M. Gabea,b, a
Physiology Unit, St. Mark’s Hospital, Watford Road, Harrow HAI 3UJ, UK Imperial College London, UK c Department of Metabolic Medicine, Hammersmith Hospital, London, UK b
Received 7 October 2005; accepted 6 December 2005
KEYWORDS Ghrelin; Peptide YY; Insulin; Appetite; Parenteral nutrition
Summary Background & aims: Patients receiving parenteral nutrition (PN) still feel hungry despite adequate provision of calories intravenously. It is not known whether PN or its constituent macronutrients acutely affect appetite and to what degree this may be mediated by ghrelin and peptide YY (PYY). Methods: Six medically stable patients (four men) with intestinal failure receiving PN received an isocaloric 200 kcal infusion on three separate occasions following a 12 h fast. The infusions consisted of either carbohydrate (10% dextrose), fat (10% intralipid) or mixed protein/carbohydrate (PN). Changes in ghrelin and peptide YY levels and changes in subjective symptoms of hunger, satiety and nausea during each macronutrient infusion were assessed. Results: None of the three infusions acutely affected subjective symptoms of hunger, satiety and nausea (P40:05 ANOVA). Ghrelin levels decreased significantly during dextrose [19.1 (35.9, 12.4), regression coefficient (95% CI), Po0:001] and parenteral nutrition infusions [18.2 (26.8, 9.6), Po0:001]. Lipid infusion had no effect on ghrelin levels but led to a significant decrease in PYY [0.076 (0.0123, 0.028), P ¼ 0:004]. Dextrose and PN infusion had no significant effect on PYY levels. Conclusions: Dextrose and PN infusions decrease ghrelin levels. Lipid infusion does not affect ghrelin levels but in contrast to oral nutrients leads to a significant
Corresponding author. Lennard-Jones Intestinal Failure Unit, St. Mark’s Hospital, Watford Road, Harrow HAI 3UJ, UK.
Tel.: +44 2082354038; fax: +44 2082354093. E-mail address: [email protected] (S.M. Gabe). 0261-5614/$ - see front matter & 2006 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2005.12.002
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decrease in PYY. Despite these changes, in patients receiving PN, macronutrient infusions do no acutely affect appetite. & 2006 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Introduction The development of home parenteral nutrition (HPN) has transformed the quality of life and indeed extended life expectancy in patients with intestinal failure. However, despite adequate replacement of calories, patients often still feel hungry, a symptom which can be particularly distressing for those minority who are unable to eat. The effects of parenteral nutrition and indeed the macronutrients within it on hunger and satiation remain relatively poorly understood. There have been multiple studies which demonstrate varying effects on food intake in animals and humans to infusions of different macronutrients.1–4 Some primate and rodent studies have demonstrated little change in oral intake despite provision of up to 60% of daily calories parenterally.5,6 Generally, however, it appears that over time infusions of parenteral nutrition, lipid, amino acids and glucose infusions will all decrease food intake.1,3,4,7 It is rare, however, for complete provision of calories to prevent food intake entirely, and hence most studies indicate an increase in overall daily energy intake as subjects continue to consume some calories orally. In acute illness it has been demonstrated that a lack of appetite is often associated with an ongoing inflammatory process, but despite this up to 89% of patients receiving parenteral nutrition in these circumstances feel hungry.8 Those undergoing long term HPN for intestinal failure (IF), however, represent a very different patient group. They are usually medically stable, though may have intermittent episodes of active disease. In a study of these patients who were not eating, Stratton and colleagues found that 75% of patients felt hungry and that 44% were distressed by this symptom. Indeed, many of the group of 16 patients reported cravings for food and 80% had a desire to eat.9,10 Most patients with HPN, however, do eat even if they do not complain of hunger. An average oral intake of around 600 kcal despite adequate parenteral caloric provision has been reported.11 The factors affecting hunger and satiation in this group are poorly understood. Parenteral infusion of macronutrients does appear to decrease food intake and appetite, but the mechanisms are unclear. The supply of nutrition by this route bypasses the cephalic phase associated with eating
and does not involve any contact of nutrients with the gastrointestinal tract. It was initially felt that much of satiety was controlled by the mechanical act of eating. We now know that energy homeostasis also involves the complex interplay of gut and brain, predominantly at a hypothalamic level, involving multiple gut-derived peptides. Two of these peptides are the orexigenic peptide ghrelin, and the anorexigenic peptide YY (PYY). Ghrelin is produced predominantly in the stomach12 with levels rising pre-prandially and falling post-prandially, suggesting a possible role in meal initiation.13 Ghrelin has been demonstrated to increase appetite and food intake in man when administered peripherally.14 Conversely, PYY is produced throughout the gut but predominantly in the Lcells of the terminal ileum and colon,15 and levels rise after eating. Peripheral administration decreases appetite and food intake in man,16 suggesting it has a physiological role as a satiety factor. The degree to which ghrelin and PYY levels are affected by different macronutrients has been investigated. Ghrelin levels are affected differentially by the different oral macronutrients. Carbohydrate and lipid meals appear to decrease ghrelin levels,17 whereas some studies demonstrate an increase in ghrelin levels following ingestion of protein or constituent amino acids.18,19 The mechanism by which this occurs is still unclear. PYY levels also show differential responses to macronutrients post-prandially, and the mechanism of this response is also unclear, though it is likely to involve both direct luminal and indirect neurohumoral processes.20,21 It is not clear whether circulating nutrients affect ghrelin, PYY and appetite. We have therefore investigated whether acute macronutrient infusions have any acute effect on subjective measures of hunger and satiation, and if so, whether any changes are associated with changes in levels of ghrelin and PYY.
Methods Patients Six medically stable patients (four men, two women, 32–73 yr, median BMI 21 kg/m2) with intestinal failure who are maintained on home
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parenteral nutrition were recruited from the outpatients department of a tertiary referral hospital. The aetiologies of intestinal failure are listed in Table 1.The study was approved by the Northwick Park and St Mark’s ethics committee (Ref: Harrow LREC 3265) and was performed in accordance with the Declaration of Helsinki. Infusions Subjects were randomly assigned to have one of three separate isocaloric 200 kcal infusions over a 2 h period on three separate study days. Infusions commenced at 10:00 after a 12 h fast. The infusions consisted of either carbohydrate (10% dextrose), fat (10% intralipid) or mixed protein/carbohydrate parenteral nutrition (PN) (median total nitrogen content 1.8 g, range 1.5–2.0 g). Subjects were continuously attached to a cardiac monitor with blood pressure and heart rate was recorded every 30 min (Dinamap, GE Medical Systems, Freiburg, Germany). Blood sampling Blood samples were drawn at 10, 0, 30, 60 and 120 min during the study. Ten milliliter samples were collected into plastic lithium heparin tubes containing 0.6 mg aprotonin. Samples were immediately centrifuged, and plasma separated and stored at 80 1C until assay. Blood glucose levels were monitored throughout using a glucometer (Medisense OptimumTM, Abbott Laboratories Ltd, Maidenhead, UK). Plasma ghrelin All samples were assayed in duplicate and in one assay to eliminate the effects of interassay variation. Ghrelin-like immunoreactivity was measured with a specific and sensitive radioimmunoassay as previously described.22 Briefly the assay cross-reacts fully (100%) with both octanoyl and des octanoyl ghrelin, but did not cross-react with any other known gastrointestinal or pancreatic hormone. The antisera (SC-10368) was obtained from Santa Cruz
Table 1
biotechnology and used at a final dilution of 1:50,000. The 125I ghrelin was prepared with Bolton & Hunter reagent (Amersham International, UK) and purified by high-pressure liquid chromatography using a linear gradient from 10% to 40% acetonitrile, 0.05% TFA over 90 min. The specific activity of ghrelin label was 48 Bq/fmol. The assay was performed in a total volume of 0.7 ml of 0.06 M phosphate buffer PH 7.2 containing 0.3% bovine serum albumin and was incubated for 3 days at 4 1C before separation of free and bound antibody label by charcoal absorption. The assay detected changes of 20 pmol/l of plasma ghrelin with a 95% confidence limit. The intra-assay coefficient of variation was 5.5%. All samples were measured in one assay to avoid inter-assay variation.
Plasma PYY All samples were assayed in duplicate and in one assay to eliminate the effects of interassay variation. PYY-like immunoreactivity was measured with a specific and sensitive radioimmunoassay, as previously described.23 The assay measured both the hormone fragment (peptide hormone YY3–36) and the full-length hormone (peptide hormone YY1–36); both are biologically active. The antiserum (Y21) was produced in a rabbit against synthetic porcine PYY (Bachem) coupled to bovine serum albumin by glutaraldehyde and used at a final dilution of 1:50,000. This antibody cross-reacts fully with the biologically active circulating forms of human PYY, but not with pancreatic polypeptide, neuropeptide Y, or other known gastrointestinal hormones. 125 I-labeled PYY was prepared by the iodogen method and purified by high-pressure liquid chromatography. The specific activity of the 125I-labeled PYY was 54 Bq/fmol. The assay was performed in a total volume of 700 ml of 0.06 M phosphate buffer, pH 7.3, containing 0.3% bovine serum albumin. The sample was incubated for 3 days at 4 1C before the separation of free and antibody-bound label by
Demographics of the patient group.
Age
Etiology of intestinal failure
Bowel anatomy
BMI
Duration PN (months)
40 40 36 73 69 32
Ischaemic bowel Crohn’s disease Crohn’s disease Ischaemic bowel Radiation enteritis Crohn’s disease
20 cm SB to stoma Colon in situ 70 cm SB to stoma Colon in situ 180 cm SB to stoma Colon in situ 30 cm to colon in continuity Entire length 65 cm to colon in continuity
28 22 20 21 19 21
12 10 14 60 56 40
None of the patients had a history of gastric surgery. SB ¼ Small bowel.
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sheep antirabbit antibody. Two hundred microliters of unextracted plasma was assayed. Two hundred microliters of PYY-free, charcoal-stripped plasma was added to standards and other reference tubes to negate any effects of non-specific assay interference. The assay detected changes of 2 pmol/l, with an intra-assay coefficient of variation of 5.8%.
presented as regression coefficients with 95% confidence intervals. VAS data were analyzed using ANOVA.
Results Glucose and insulin Both PN and dextrose infusions led to a significant increase in glucose and insulin levels (Table 2 and 3). Lipid infusion had no significant effect on either glucose or insulin levels.
Plasma insulin Insulin was measured using commercial immunoassays (Abbott, UK) with an intra-assay coefficient of variation of 4.6%.
Ghrelin levels Baseline ghrelin levels did not differ significantly between the three infusion days (Fig. 1). Ghrelin
Ghrelin (pmol/l)
Symptom scores Subjective assessment of hunger, satiety and nausea was made before and throughout each infusion with standardized description anchored visual analogue scores.24
Statistics Statistical analysis was carried out using Stata software (version 7.0. Stata Corporation, College Station, Texas, US). Linear regression analysis was used to examine the effect of each infusion on the different outcome measures over time. Included in the model were adjustments for patient. Glucose, PYY and insulin levels were all found to have a skewed distribution, and were given a log transformation. Linear regression was also used to examine the effect of glucose and insulin on ghrelin and PYY levels. The models included adjustments for patient, time and treatment. These data are
Table 2 Time
PN Lipid Dextrose
1400 1300 1200 1100 1000 900 800 700 600 500
PN
0
Lipid
30
Dextrose
60
90
120
Time (minutes)
Figure 1 Changes in ghrelin levels during PN, dextrose and lipid infusions. Linear regression analysis reveals a significant decrease in ghrelin levels over time during PN [18.2 (26.8, 9.6), regression coefficient (95% CI), Po0:001] and dextrose [19.1 (25.9, 12.4), Po0:001] infusions.
Glucose levels during each of the three infusions. Glucose mmol/l 0
30
60
120
6.670.4 6.470.4 6.670.4
9.070.9 6.370.4 9.471.4
9.771.2 6.170.3 10.171.7
9.171.1 6.570.4 11.271.6
Regression coefficient (95% Cl)
P-value
0.078 (0.047, 0.109) 0.002 (0.003, 0.006) 0.089 (0.053, 0.124)
o0.001 0.52 o0.001
Regression coefficient (95% Cl)
P-value
0.71 (0.39, 1.03) 0.022 (0.005, 0.049) 0.391 (0.255, 0.528)
o0.001 0.10 o0.001
Glucose levels rose significantly during PN and dextrose infusions.
Table 3 Time
PN Lipid Dextrose
Insulin levels during each of the three infusions. Insulin mU/I 0
30
60
120
3.570.8 4.272 4.371.3
48.5718 5.472.1 19.677
66723 4.871.9 20.474
51.9735 5.672.2 23.876.9
Insulin levels rose significantly during PN and dextrose infusions.
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C.D. Murray et al. 80 70 60
VAS (mm)
levels decreased significantly during dextrose [19.1 (35.9, 12.4), regression coefficient (95% CI), Po0:001] and parenteral nutrition infusions [18.2 (26.8, 9.6), Po0:001]. Lipid infusion had no effect on ghrelin levels [1.3 (10.8, 8.2), P ¼ 0:78]. Linear regression analysis indicated a significant effect of glucose levels on ghrelin levels during PN infusion only. During PN infusion there was a significant decrease in ghrelin associated with an increase in blood glucose [210 (411, 9), P ¼ 0:04]. There were no significant relationships between insulin increase and fall in ghrelin levels during any of the infusions [31 (66, 4), P ¼ 0:08].
50 40 30 20 10 0 0
30
60
90
120
Time (minutes)
(A) 80 70
Symptom scores There were no significant changes in VAS scores for any of the measurable symptoms of hunger, satiety or nausea during the three different infusions (Fig. 3A, B and C, respectively) (P40:05, ANOVA).
50 40 30 20 10 0 0
30
60
90
120
90
120
Time (minutes)
(B) 50
VAS (mm)
PYY levels Baseline PYY levels did not differ significantly between the three infusion days (Fig. 2). PYY levels only decreased significantly during lipid infusion [0.076 (0.0123, 0.028), P ¼ 0:004]. Dextrose and PN infusion had no significant effect on PYY levels. During lipid infusion only, there was a significant decrease in PYY associated with an increase in insulin [0.26 (0.45, 0.06), P ¼ 0:01].
VAS (mm)
60
25
0 0
(C)
Discussion We demonstrated that the infusion of standard carbohydrate/protein parenteral nutrition, lipid and dextrose had no effect on acute subjective
35
Lipid
PN
Dextrose
PYY (pmol/l)
30 25 20 15 10 0
30
60
90
120
Time (minutes)
Figure 2 Changes in PYY levels during PN, dextrose and lipid infusions. PYY levels decreased significantly during lipid infusion only [0.076 (0.0123, 0.028), regression coefficient (95% CI), P ¼ 0:004].
30
60
Time (minutes)
Figure 3 The effects of PN ( ), lipid ( ) and dextrose ( ) infusions on hunger (A), satiety (B) and nausea (C) VAS scores. There were no significant differences in symptoms throughout each infusion (P40:05, ANOVA).
feelings of hunger and satiation in a group of patients with intestinal failure. We also showed a decrease in ghrelin levels during parenteral nutrition and dextrose infusion, and a marked decrease in PYY levels during lipid infusion. These changes in peptide levels were, however, not associated with changes in appetite. Previous studies have not examined the acute effects of macronutrients on hunger and satiation, although most studies do demonstrate a decrease in food intake over time after commencement of parenteral nutrition. The mechanisms controlling energy homeostasis are clearly complex. Recent studies have demonstrated that gut derived peptides are important for the short and long term
ARTICLE IN PRESS Effect of macronutrient infusion on ghrelin and PYY control of food intake and both ghrelin14,25 and PYY16 appear to play important and opposing roles. The effects of ghrelin appear to be mediated via the neuropeptide-Y neurons in the arcuate nucleus of the hypothalamus, as blockade of the NPY-Y1 receptor blocks this effect.26 It is not entirely clear whether the orexigenic action of ghrelin is mediated directly at a central level or by acting in the periphery by its action on the vagus nerve.24,27 In addition, it is not clear what mechanisms control the fluctuating levels of ghrelin. There are likely to be several mechanisms that lead to the post-prandial fall in ghrelin levels associated with satiation. Several studies have proposed that ghrelin levels are controlled reciprocally by insulin levels, which is supported by findings demonstrating a fall in ghrelin levels during euglycaemic hyerinsulinaemia.28–31 Fasting ghrelin levels also correlate negatively with insulin resistance and insulin levels in obese patients,32 again suggesting insulin to have an important role. Supraphysiological hyperglycaemia decreases ghrelin levels33; however, it remains unclear whether changes in glucose or insulin are the main factors controlling ghrelin levels. It has been reported that supraphysiological levels of insulin are required to decreases ghrelin concentrations,34 and similarly that when intravenous ghrelin and bolus insulin were given together there was no effect on ghrelin levels.35 In our study ghrelin levels fell markedly during infusion of either PN or dextrose infusions. The PN in this study was predominantly dextrose based, so the fall in ghrelin levels is perhaps not surprising. For both infusions there was a significant increase in glucose and insulin levels. Despite this, linear regression analysis reveals there only to be a significant decrease in ghrelin associated with an increase in glucose during the dextrose infusion. Glucose levels were higher during this infusion, and at times outside the physiological range. This supports the theory that hyperglycaemia is a determinant of ghrelin levels only at supraphysiological levels. In our study there was a suggestion of an effect of insulin on ghrelin levels when all the infusions are considered together, but this did not reach significance ðP ¼ 0:08Þ. We confirmed previous findings that lipid infusion had no effect on ghrelin levels.36 Lipid infusion also had little effect on glucose and insulin levels. Mohlig et al. demonstrated similar findings after infusion of intralipid-heparin, although addition of insulin in this study did lead to a decrease in ghrelin levels, suggesting again that insulin is important.36 This is in contrast to the effects of oral lipids which have been demonstrated in most17 but not all
631 studies37 to lead to a decrease in ghrelin. This suggests that the decrease in ghrelin levels seen after a lipid meal is not secondary to circulating nutrients, but rather to other luminal or neurohumoral factors. Ghrelin levels have been demonstrated to be lower in patients with short bowel syndrome, presumably secondary to a loss of ghrelin secreting cells,38 but to date there have been no studies looking at the dynamic response to oral challenge in this heterogenous group. This study did not involve an oral challenge so the responses were not secondary luminal nutrient contact. The group is heterogenous, both in terms of aetiology of intestinal failure and indeed in BMI. Despite the difference in BMI the fasting levels of ghrelin and PYY were not significantly different between patients, and the reported significant responses in peptide levels were not related to BMI. None of the patients were nil by mouth, except for the study day, and all of the patients therefore had regular nutrient contact with the upper gastrointestinal tract. This is important, especially in terms of ghrelin responses since at least 80% of ghrelin production is from the stomach with the majority of the rest being produced in the proximal gut. Therefore, despite the varying aetiologies of intestinal failure, one would not expect significant upper gut adaptation or atrophy to be significantly different between patients. The differing anatomy of the six subjects in this study is more relevant to the PYY responses in this study. PYY is produced in enteroendocrine cells throughout the gut, but is predominantly produced in the terminal ileum and colon, with levels being highest in the rectum.15. In patients with short bowel syndrome with bowel in continuity (i.e. loss of a large amount of small bowel but intact colon), PYY levels are high and they demonstrate exaggerated responses to feeding, something that has been proposed to be associated with the ‘colonic brake’ as PYY delays gastric emptying.39 Only two of our subjects fit this description, with one demonstrating high levels of PYY. Three further subjects had jejunostomies with the colon in situ but out of continuity, and one subject had radiation enteritis. Despite the different aetiologies of their intestinal failure, all subjects had PYY levels within the normal fasting range. The increase in PYY levels post-prandially appears to be controlled by both direct luminal stimuli and also indirect neuro-humoral mechanisms.21,40 A study in dogs did not demonstrate any change in PYY levels following amino acid infusion,40 but otherwise the effects of intravenous macronutrients on PYY levels are not known.
ARTICLE IN PRESS 632 In our study, PYY levels fell during each of the infusions, something that would be expected as the period of fast lengthened. However, the degree to which the levels fell was significant during the lipid infusion. Interestingly it appears that during lipid infusion, the degree to which PYY levels decrease is related to the level of insulin. This may suggest that whereas luminal contact in the proximal and distal gut appears to lead to PYY release by different mechanisms, micelle absorption into the circulation actually has an inhibitory effect on PYY release, possibly related to an increase in insulin resistance. We should, however, interpret these results with care given that this is a small group with heterogenous bowel anatomy. What is clear though is that none of the infusions led to an increase in PYY, suggesting that neither the direct nor the indirect mechanisms associated with postprandial increase in PYY levels are associated with post-absorptive nutrient circulation. Despite the significant changes in peptide levels during dextrose, lipid and PN infusions, we did not demonstrate any acute subjective changes in symptoms of appetite and satiety. This may suggest that at least acutely, the changes in ghrelin and PYY levels do not play an important role in this group of patients, and that, for example, ghrelin antagonists or PYY agonists may not be viable targets for the treatment of distressing symptoms of hunger in these patients. However, more studies are required to investigate whether changes in these peptides following regular macronutrient infusions are related to changes in caloric intake over time. The mechanisms controlling hunger and satiation in patients undergoing home parenteral nutrition are not clearly understood. However, we have demonstrated that in contrast to orally consumed nutrients there is no acute change in subjective reporting of these symptoms during infusions of different macronutrients. Despite this ghrelin levels fall in response to dextrose and carbohydrate/protein standard parenteral nutrition, which suggests the post-prandial fall in ghrelin levels may not on its own be sufficient as a satiety factor without luminal nutrient involvement. Parenteral macronutrients also do not increase PYY levels and lipid infusions appear to decrease PYY in this group of patients with intestinal failure.
Acknowledgments We would like to thank Clinical Nurse Specialists Debbie Jones, Sally Crowther and Angie Davidson
C.D. Murray et al. for their help with this study. CLR is a Wellcome Clinical Research Training Fellow.
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ORIGINAL ARTICLE
The effect of different macronutrient infusions on appetite, ghrelin and peptide YY in parenterally fed patients Charles D. Murraya,b, Carel W. le Rouxb,c, Catherine Gouveiab, Paul Bassetta, Mohammad A. Ghateib,c, Stephen R. Bloomb,c, Anton V. Emmanuela,b, Simon M. Gabea,b, a
Physiology Unit, St. Mark’s Hospital, Watford Road, Harrow HAI 3UJ, UK Imperial College London, UK c Department of Metabolic Medicine, Hammersmith Hospital, London, UK b
Received 7 October 2005; accepted 6 December 2005
KEYWORDS Ghrelin; Peptide YY; Insulin; Appetite; Parenteral nutrition
Summary Background & aims: Patients receiving parenteral nutrition (PN) still feel hungry despite adequate provision of calories intravenously. It is not known whether PN or its constituent macronutrients acutely affect appetite and to what degree this may be mediated by ghrelin and peptide YY (PYY). Methods: Six medically stable patients (four men) with intestinal failure receiving PN received an isocaloric 200 kcal infusion on three separate occasions following a 12 h fast. The infusions consisted of either carbohydrate (10% dextrose), fat (10% intralipid) or mixed protein/carbohydrate (PN). Changes in ghrelin and peptide YY levels and changes in subjective symptoms of hunger, satiety and nausea during each macronutrient infusion were assessed. Results: None of the three infusions acutely affected subjective symptoms of hunger, satiety and nausea (P40:05 ANOVA). Ghrelin levels decreased significantly during dextrose [19.1 (35.9, 12.4), regression coefficient (95% CI), Po0:001] and parenteral nutrition infusions [18.2 (26.8, 9.6), Po0:001]. Lipid infusion had no effect on ghrelin levels but led to a significant decrease in PYY [0.076 (0.0123, 0.028), P ¼ 0:004]. Dextrose and PN infusion had no significant effect on PYY levels. Conclusions: Dextrose and PN infusions decrease ghrelin levels. Lipid infusion does not affect ghrelin levels but in contrast to oral nutrients leads to a significant
Corresponding author. Lennard-Jones Intestinal Failure Unit, St. Mark’s Hospital, Watford Road, Harrow HAI 3UJ, UK.
Tel.: +44 2082354038; fax: +44 2082354093. E-mail address: [email protected] (S.M. Gabe). 0261-5614/$ - see front matter & 2006 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2005.12.002
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decrease in PYY. Despite these changes, in patients receiving PN, macronutrient infusions do no acutely affect appetite. & 2006 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Introduction The development of home parenteral nutrition (HPN) has transformed the quality of life and indeed extended life expectancy in patients with intestinal failure. However, despite adequate replacement of calories, patients often still feel hungry, a symptom which can be particularly distressing for those minority who are unable to eat. The effects of parenteral nutrition and indeed the macronutrients within it on hunger and satiation remain relatively poorly understood. There have been multiple studies which demonstrate varying effects on food intake in animals and humans to infusions of different macronutrients.1–4 Some primate and rodent studies have demonstrated little change in oral intake despite provision of up to 60% of daily calories parenterally.5,6 Generally, however, it appears that over time infusions of parenteral nutrition, lipid, amino acids and glucose infusions will all decrease food intake.1,3,4,7 It is rare, however, for complete provision of calories to prevent food intake entirely, and hence most studies indicate an increase in overall daily energy intake as subjects continue to consume some calories orally. In acute illness it has been demonstrated that a lack of appetite is often associated with an ongoing inflammatory process, but despite this up to 89% of patients receiving parenteral nutrition in these circumstances feel hungry.8 Those undergoing long term HPN for intestinal failure (IF), however, represent a very different patient group. They are usually medically stable, though may have intermittent episodes of active disease. In a study of these patients who were not eating, Stratton and colleagues found that 75% of patients felt hungry and that 44% were distressed by this symptom. Indeed, many of the group of 16 patients reported cravings for food and 80% had a desire to eat.9,10 Most patients with HPN, however, do eat even if they do not complain of hunger. An average oral intake of around 600 kcal despite adequate parenteral caloric provision has been reported.11 The factors affecting hunger and satiation in this group are poorly understood. Parenteral infusion of macronutrients does appear to decrease food intake and appetite, but the mechanisms are unclear. The supply of nutrition by this route bypasses the cephalic phase associated with eating
and does not involve any contact of nutrients with the gastrointestinal tract. It was initially felt that much of satiety was controlled by the mechanical act of eating. We now know that energy homeostasis also involves the complex interplay of gut and brain, predominantly at a hypothalamic level, involving multiple gut-derived peptides. Two of these peptides are the orexigenic peptide ghrelin, and the anorexigenic peptide YY (PYY). Ghrelin is produced predominantly in the stomach12 with levels rising pre-prandially and falling post-prandially, suggesting a possible role in meal initiation.13 Ghrelin has been demonstrated to increase appetite and food intake in man when administered peripherally.14 Conversely, PYY is produced throughout the gut but predominantly in the Lcells of the terminal ileum and colon,15 and levels rise after eating. Peripheral administration decreases appetite and food intake in man,16 suggesting it has a physiological role as a satiety factor. The degree to which ghrelin and PYY levels are affected by different macronutrients has been investigated. Ghrelin levels are affected differentially by the different oral macronutrients. Carbohydrate and lipid meals appear to decrease ghrelin levels,17 whereas some studies demonstrate an increase in ghrelin levels following ingestion of protein or constituent amino acids.18,19 The mechanism by which this occurs is still unclear. PYY levels also show differential responses to macronutrients post-prandially, and the mechanism of this response is also unclear, though it is likely to involve both direct luminal and indirect neurohumoral processes.20,21 It is not clear whether circulating nutrients affect ghrelin, PYY and appetite. We have therefore investigated whether acute macronutrient infusions have any acute effect on subjective measures of hunger and satiation, and if so, whether any changes are associated with changes in levels of ghrelin and PYY.
Methods Patients Six medically stable patients (four men, two women, 32–73 yr, median BMI 21 kg/m2) with intestinal failure who are maintained on home
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parenteral nutrition were recruited from the outpatients department of a tertiary referral hospital. The aetiologies of intestinal failure are listed in Table 1.The study was approved by the Northwick Park and St Mark’s ethics committee (Ref: Harrow LREC 3265) and was performed in accordance with the Declaration of Helsinki. Infusions Subjects were randomly assigned to have one of three separate isocaloric 200 kcal infusions over a 2 h period on three separate study days. Infusions commenced at 10:00 after a 12 h fast. The infusions consisted of either carbohydrate (10% dextrose), fat (10% intralipid) or mixed protein/carbohydrate parenteral nutrition (PN) (median total nitrogen content 1.8 g, range 1.5–2.0 g). Subjects were continuously attached to a cardiac monitor with blood pressure and heart rate was recorded every 30 min (Dinamap, GE Medical Systems, Freiburg, Germany). Blood sampling Blood samples were drawn at 10, 0, 30, 60 and 120 min during the study. Ten milliliter samples were collected into plastic lithium heparin tubes containing 0.6 mg aprotonin. Samples were immediately centrifuged, and plasma separated and stored at 80 1C until assay. Blood glucose levels were monitored throughout using a glucometer (Medisense OptimumTM, Abbott Laboratories Ltd, Maidenhead, UK). Plasma ghrelin All samples were assayed in duplicate and in one assay to eliminate the effects of interassay variation. Ghrelin-like immunoreactivity was measured with a specific and sensitive radioimmunoassay as previously described.22 Briefly the assay cross-reacts fully (100%) with both octanoyl and des octanoyl ghrelin, but did not cross-react with any other known gastrointestinal or pancreatic hormone. The antisera (SC-10368) was obtained from Santa Cruz
Table 1
biotechnology and used at a final dilution of 1:50,000. The 125I ghrelin was prepared with Bolton & Hunter reagent (Amersham International, UK) and purified by high-pressure liquid chromatography using a linear gradient from 10% to 40% acetonitrile, 0.05% TFA over 90 min. The specific activity of ghrelin label was 48 Bq/fmol. The assay was performed in a total volume of 0.7 ml of 0.06 M phosphate buffer PH 7.2 containing 0.3% bovine serum albumin and was incubated for 3 days at 4 1C before separation of free and bound antibody label by charcoal absorption. The assay detected changes of 20 pmol/l of plasma ghrelin with a 95% confidence limit. The intra-assay coefficient of variation was 5.5%. All samples were measured in one assay to avoid inter-assay variation.
Plasma PYY All samples were assayed in duplicate and in one assay to eliminate the effects of interassay variation. PYY-like immunoreactivity was measured with a specific and sensitive radioimmunoassay, as previously described.23 The assay measured both the hormone fragment (peptide hormone YY3–36) and the full-length hormone (peptide hormone YY1–36); both are biologically active. The antiserum (Y21) was produced in a rabbit against synthetic porcine PYY (Bachem) coupled to bovine serum albumin by glutaraldehyde and used at a final dilution of 1:50,000. This antibody cross-reacts fully with the biologically active circulating forms of human PYY, but not with pancreatic polypeptide, neuropeptide Y, or other known gastrointestinal hormones. 125 I-labeled PYY was prepared by the iodogen method and purified by high-pressure liquid chromatography. The specific activity of the 125I-labeled PYY was 54 Bq/fmol. The assay was performed in a total volume of 700 ml of 0.06 M phosphate buffer, pH 7.3, containing 0.3% bovine serum albumin. The sample was incubated for 3 days at 4 1C before the separation of free and antibody-bound label by
Demographics of the patient group.
Age
Etiology of intestinal failure
Bowel anatomy
BMI
Duration PN (months)
40 40 36 73 69 32
Ischaemic bowel Crohn’s disease Crohn’s disease Ischaemic bowel Radiation enteritis Crohn’s disease
20 cm SB to stoma Colon in situ 70 cm SB to stoma Colon in situ 180 cm SB to stoma Colon in situ 30 cm to colon in continuity Entire length 65 cm to colon in continuity
28 22 20 21 19 21
12 10 14 60 56 40
None of the patients had a history of gastric surgery. SB ¼ Small bowel.
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sheep antirabbit antibody. Two hundred microliters of unextracted plasma was assayed. Two hundred microliters of PYY-free, charcoal-stripped plasma was added to standards and other reference tubes to negate any effects of non-specific assay interference. The assay detected changes of 2 pmol/l, with an intra-assay coefficient of variation of 5.8%.
presented as regression coefficients with 95% confidence intervals. VAS data were analyzed using ANOVA.
Results Glucose and insulin Both PN and dextrose infusions led to a significant increase in glucose and insulin levels (Table 2 and 3). Lipid infusion had no significant effect on either glucose or insulin levels.
Plasma insulin Insulin was measured using commercial immunoassays (Abbott, UK) with an intra-assay coefficient of variation of 4.6%.
Ghrelin levels Baseline ghrelin levels did not differ significantly between the three infusion days (Fig. 1). Ghrelin
Ghrelin (pmol/l)
Symptom scores Subjective assessment of hunger, satiety and nausea was made before and throughout each infusion with standardized description anchored visual analogue scores.24
Statistics Statistical analysis was carried out using Stata software (version 7.0. Stata Corporation, College Station, Texas, US). Linear regression analysis was used to examine the effect of each infusion on the different outcome measures over time. Included in the model were adjustments for patient. Glucose, PYY and insulin levels were all found to have a skewed distribution, and were given a log transformation. Linear regression was also used to examine the effect of glucose and insulin on ghrelin and PYY levels. The models included adjustments for patient, time and treatment. These data are
Table 2 Time
PN Lipid Dextrose
1400 1300 1200 1100 1000 900 800 700 600 500
PN
0
Lipid
30
Dextrose
60
90
120
Time (minutes)
Figure 1 Changes in ghrelin levels during PN, dextrose and lipid infusions. Linear regression analysis reveals a significant decrease in ghrelin levels over time during PN [18.2 (26.8, 9.6), regression coefficient (95% CI), Po0:001] and dextrose [19.1 (25.9, 12.4), Po0:001] infusions.
Glucose levels during each of the three infusions. Glucose mmol/l 0
30
60
120
6.670.4 6.470.4 6.670.4
9.070.9 6.370.4 9.471.4
9.771.2 6.170.3 10.171.7
9.171.1 6.570.4 11.271.6
Regression coefficient (95% Cl)
P-value
0.078 (0.047, 0.109) 0.002 (0.003, 0.006) 0.089 (0.053, 0.124)
o0.001 0.52 o0.001
Regression coefficient (95% Cl)
P-value
0.71 (0.39, 1.03) 0.022 (0.005, 0.049) 0.391 (0.255, 0.528)
o0.001 0.10 o0.001
Glucose levels rose significantly during PN and dextrose infusions.
Table 3 Time
PN Lipid Dextrose
Insulin levels during each of the three infusions. Insulin mU/I 0
30
60
120
3.570.8 4.272 4.371.3
48.5718 5.472.1 19.677
66723 4.871.9 20.474
51.9735 5.672.2 23.876.9
Insulin levels rose significantly during PN and dextrose infusions.
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C.D. Murray et al. 80 70 60
VAS (mm)
levels decreased significantly during dextrose [19.1 (35.9, 12.4), regression coefficient (95% CI), Po0:001] and parenteral nutrition infusions [18.2 (26.8, 9.6), Po0:001]. Lipid infusion had no effect on ghrelin levels [1.3 (10.8, 8.2), P ¼ 0:78]. Linear regression analysis indicated a significant effect of glucose levels on ghrelin levels during PN infusion only. During PN infusion there was a significant decrease in ghrelin associated with an increase in blood glucose [210 (411, 9), P ¼ 0:04]. There were no significant relationships between insulin increase and fall in ghrelin levels during any of the infusions [31 (66, 4), P ¼ 0:08].
50 40 30 20 10 0 0
30
60
90
120
Time (minutes)
(A) 80 70
Symptom scores There were no significant changes in VAS scores for any of the measurable symptoms of hunger, satiety or nausea during the three different infusions (Fig. 3A, B and C, respectively) (P40:05, ANOVA).
50 40 30 20 10 0 0
30
60
90
120
90
120
Time (minutes)
(B) 50
VAS (mm)
PYY levels Baseline PYY levels did not differ significantly between the three infusion days (Fig. 2). PYY levels only decreased significantly during lipid infusion [0.076 (0.0123, 0.028), P ¼ 0:004]. Dextrose and PN infusion had no significant effect on PYY levels. During lipid infusion only, there was a significant decrease in PYY associated with an increase in insulin [0.26 (0.45, 0.06), P ¼ 0:01].
VAS (mm)
60
25
0 0
(C)
Discussion We demonstrated that the infusion of standard carbohydrate/protein parenteral nutrition, lipid and dextrose had no effect on acute subjective
35
Lipid
PN
Dextrose
PYY (pmol/l)
30 25 20 15 10 0
30
60
90
120
Time (minutes)
Figure 2 Changes in PYY levels during PN, dextrose and lipid infusions. PYY levels decreased significantly during lipid infusion only [0.076 (0.0123, 0.028), regression coefficient (95% CI), P ¼ 0:004].
30
60
Time (minutes)
Figure 3 The effects of PN ( ), lipid ( ) and dextrose ( ) infusions on hunger (A), satiety (B) and nausea (C) VAS scores. There were no significant differences in symptoms throughout each infusion (P40:05, ANOVA).
feelings of hunger and satiation in a group of patients with intestinal failure. We also showed a decrease in ghrelin levels during parenteral nutrition and dextrose infusion, and a marked decrease in PYY levels during lipid infusion. These changes in peptide levels were, however, not associated with changes in appetite. Previous studies have not examined the acute effects of macronutrients on hunger and satiation, although most studies do demonstrate a decrease in food intake over time after commencement of parenteral nutrition. The mechanisms controlling energy homeostasis are clearly complex. Recent studies have demonstrated that gut derived peptides are important for the short and long term
ARTICLE IN PRESS Effect of macronutrient infusion on ghrelin and PYY control of food intake and both ghrelin14,25 and PYY16 appear to play important and opposing roles. The effects of ghrelin appear to be mediated via the neuropeptide-Y neurons in the arcuate nucleus of the hypothalamus, as blockade of the NPY-Y1 receptor blocks this effect.26 It is not entirely clear whether the orexigenic action of ghrelin is mediated directly at a central level or by acting in the periphery by its action on the vagus nerve.24,27 In addition, it is not clear what mechanisms control the fluctuating levels of ghrelin. There are likely to be several mechanisms that lead to the post-prandial fall in ghrelin levels associated with satiation. Several studies have proposed that ghrelin levels are controlled reciprocally by insulin levels, which is supported by findings demonstrating a fall in ghrelin levels during euglycaemic hyerinsulinaemia.28–31 Fasting ghrelin levels also correlate negatively with insulin resistance and insulin levels in obese patients,32 again suggesting insulin to have an important role. Supraphysiological hyperglycaemia decreases ghrelin levels33; however, it remains unclear whether changes in glucose or insulin are the main factors controlling ghrelin levels. It has been reported that supraphysiological levels of insulin are required to decreases ghrelin concentrations,34 and similarly that when intravenous ghrelin and bolus insulin were given together there was no effect on ghrelin levels.35 In our study ghrelin levels fell markedly during infusion of either PN or dextrose infusions. The PN in this study was predominantly dextrose based, so the fall in ghrelin levels is perhaps not surprising. For both infusions there was a significant increase in glucose and insulin levels. Despite this, linear regression analysis reveals there only to be a significant decrease in ghrelin associated with an increase in glucose during the dextrose infusion. Glucose levels were higher during this infusion, and at times outside the physiological range. This supports the theory that hyperglycaemia is a determinant of ghrelin levels only at supraphysiological levels. In our study there was a suggestion of an effect of insulin on ghrelin levels when all the infusions are considered together, but this did not reach significance ðP ¼ 0:08Þ. We confirmed previous findings that lipid infusion had no effect on ghrelin levels.36 Lipid infusion also had little effect on glucose and insulin levels. Mohlig et al. demonstrated similar findings after infusion of intralipid-heparin, although addition of insulin in this study did lead to a decrease in ghrelin levels, suggesting again that insulin is important.36 This is in contrast to the effects of oral lipids which have been demonstrated in most17 but not all
631 studies37 to lead to a decrease in ghrelin. This suggests that the decrease in ghrelin levels seen after a lipid meal is not secondary to circulating nutrients, but rather to other luminal or neurohumoral factors. Ghrelin levels have been demonstrated to be lower in patients with short bowel syndrome, presumably secondary to a loss of ghrelin secreting cells,38 but to date there have been no studies looking at the dynamic response to oral challenge in this heterogenous group. This study did not involve an oral challenge so the responses were not secondary luminal nutrient contact. The group is heterogenous, both in terms of aetiology of intestinal failure and indeed in BMI. Despite the difference in BMI the fasting levels of ghrelin and PYY were not significantly different between patients, and the reported significant responses in peptide levels were not related to BMI. None of the patients were nil by mouth, except for the study day, and all of the patients therefore had regular nutrient contact with the upper gastrointestinal tract. This is important, especially in terms of ghrelin responses since at least 80% of ghrelin production is from the stomach with the majority of the rest being produced in the proximal gut. Therefore, despite the varying aetiologies of intestinal failure, one would not expect significant upper gut adaptation or atrophy to be significantly different between patients. The differing anatomy of the six subjects in this study is more relevant to the PYY responses in this study. PYY is produced in enteroendocrine cells throughout the gut, but is predominantly produced in the terminal ileum and colon, with levels being highest in the rectum.15. In patients with short bowel syndrome with bowel in continuity (i.e. loss of a large amount of small bowel but intact colon), PYY levels are high and they demonstrate exaggerated responses to feeding, something that has been proposed to be associated with the ‘colonic brake’ as PYY delays gastric emptying.39 Only two of our subjects fit this description, with one demonstrating high levels of PYY. Three further subjects had jejunostomies with the colon in situ but out of continuity, and one subject had radiation enteritis. Despite the different aetiologies of their intestinal failure, all subjects had PYY levels within the normal fasting range. The increase in PYY levels post-prandially appears to be controlled by both direct luminal stimuli and also indirect neuro-humoral mechanisms.21,40 A study in dogs did not demonstrate any change in PYY levels following amino acid infusion,40 but otherwise the effects of intravenous macronutrients on PYY levels are not known.
ARTICLE IN PRESS 632 In our study, PYY levels fell during each of the infusions, something that would be expected as the period of fast lengthened. However, the degree to which the levels fell was significant during the lipid infusion. Interestingly it appears that during lipid infusion, the degree to which PYY levels decrease is related to the level of insulin. This may suggest that whereas luminal contact in the proximal and distal gut appears to lead to PYY release by different mechanisms, micelle absorption into the circulation actually has an inhibitory effect on PYY release, possibly related to an increase in insulin resistance. We should, however, interpret these results with care given that this is a small group with heterogenous bowel anatomy. What is clear though is that none of the infusions led to an increase in PYY, suggesting that neither the direct nor the indirect mechanisms associated with postprandial increase in PYY levels are associated with post-absorptive nutrient circulation. Despite the significant changes in peptide levels during dextrose, lipid and PN infusions, we did not demonstrate any acute subjective changes in symptoms of appetite and satiety. This may suggest that at least acutely, the changes in ghrelin and PYY levels do not play an important role in this group of patients, and that, for example, ghrelin antagonists or PYY agonists may not be viable targets for the treatment of distressing symptoms of hunger in these patients. However, more studies are required to investigate whether changes in these peptides following regular macronutrient infusions are related to changes in caloric intake over time. The mechanisms controlling hunger and satiation in patients undergoing home parenteral nutrition are not clearly understood. However, we have demonstrated that in contrast to orally consumed nutrients there is no acute change in subjective reporting of these symptoms during infusions of different macronutrients. Despite this ghrelin levels fall in response to dextrose and carbohydrate/protein standard parenteral nutrition, which suggests the post-prandial fall in ghrelin levels may not on its own be sufficient as a satiety factor without luminal nutrient involvement. Parenteral macronutrients also do not increase PYY levels and lipid infusions appear to decrease PYY in this group of patients with intestinal failure.
Acknowledgments We would like to thank Clinical Nurse Specialists Debbie Jones, Sally Crowther and Angie Davidson
C.D. Murray et al. for their help with this study. CLR is a Wellcome Clinical Research Training Fellow.
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