Conditions: Healthy Volunteers
Keywords: Deiodinase Bile Acids Cholecystokinin Thyroid Hormones Energy Expenditure Healthy Volunteer
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 31
Healthy volunteers: no

Brief summary:
Postprandial thermogenesis, or thermic effect of food are terms that describe the increase in utilization of energy by the human body following a meal. The mechanisms involved in this process are believed to differ according to the type of food consumed, whether fat, protein or carbohydrate.

The bile acids (BAs), unique substances secreted by the gall bladder into the gut after a meal, play an important role in the absorption of fat and the management of cholesterol stores in the body. Recent studies suggest that BAs may also serve as regulators of energy expenditure (consumption) in the cells of our body by increasing the production of T3, an active form of thyroid hormone. T3 in turn is believed to increase the efficiency with which our bodies burn calories thereby generating heat. Although this process has been shown to be effective in rodents who demonstrated weight loss after treatment, the role of BAs in humans is poorly understood. Thus we do not know whether endogenous (produced by the body) or exogenous (taken as medication) BAs play a significant role in the maintenance of body weight. We hypothesize that, similarly to rodents, humans will respond to BAs by increasing energy expenditure via the production of the active form of thyroid hormone.

This randomized, cross-over study will look at changes in thyroid hormones and energy consumption in response to stimuli of endogenous BA secretion including dietary content, and to the intake of pharmacological doses of bile acids.

Following a two-day period of equilibration diet, 30 healthy volunteers will be randomly assigned to receive either a high-fat or high-carbohydrate isocaloric meal followed by a 6-hour metabolic chamber stay; the next day they will be crossed-over to the alternate intervention. During the following three days, the study subjects will again be randomized to receive either an intravenous injection of sincalide (the C-terminal octapeptide fragment of cholecystokinin) 0.04 mcg/kg or placebo and P.O. placebo, or I.V. placebo and 15 mg/kg of BA (ursodiol) with similar metabolic chamber stays and cross-over design.

The data gathered from this study will provide greater insight into the physiological and molecular mechanism(s) regulating the relation between endogenous bile acid secretion and energy metabolism in response to meals, as well as the role of BAs per se on energy metabolism.

Detailed description:
Postprandial thermogenesis, or thermic effect of food are terms that describe the increase in utilization of energy by the human body following a meal. The mechanisms involved in this process are believed to differ according to the type of food consumed, whether fat, protein or carbohydrate.

The bile acids (BAs), unique substances secreted by the gall bladder into the gut after a meal, play an important role in the absorption of fat and the management of cholesterol stores in the body. Recent studies suggest that BAs may also serve as regulators of energy expenditure (consumption) in the cells of our body by increasing the production of T3, an active form of thyroid hormone. T3 in turn is believed to increase the efficiency with which our bodies burn calories thereby generating heat. Although this process has been shown to be effective in rodents who demonstrated weight loss after treatment, the role of BAs in humans is poorly understood. Thus we do not know whether endogenous (produced by the body) or exogenous (taken as medication) BAs play a significant role in the maintenance of body weight. We hypothesize that, similarly to rodents, humans will respond to BAs by increasing energy expenditure via the production of the active form of thyroid hormone.

This randomized, cross-over study will look at changes in thyroid hormones and energy consumption in response to stimuli of endogenous BA secretion including dietary content, and to the intake of pharmacological doses of bile acids.

Following a two-day period of equilibration diet, 30 healthy volunteers will be randomly assigned to receive either a high-fat or high-carbohydrate isocaloric meal followed by a 6-hour metabolic chamber stay; the next day they will be crossed-over to the alternate intervention. During the following three days, the study subjects will again be randomized to receive either an intravenous injection of sincalide (the C-terminal octapeptide fragment of cholecystokinin) 0.04 mcg/kg or placebo and P.O. placebo, or I.V. placebo and 15 mg/kg of BA (ursodiol) with similar metabolic chamber stays and cross-over design.

The following parameters will be recorded and compared to placebo:

Energy expenditure

Substrate utilization

Spontaneous movements

Skin and core temperature

Serial changes in circulating thyroid hormones

Serial changes in bile acid serum concentrations

The data gathered from this study will provide greater insight into the physiological and molecular mechanism(s) regulating the relation between endogenous bile acid secretion and energy metabolism in response to meals, as well as the role of BAs per se on energy metabolism.

Arms:
arm 1: Participants randomized to consume a 100% carbohydrate meal day 1, then a high fat (72% fat, 8% protein, 20% carbohydrate) meal day 2, IV sincalide 0.04mcg/kg + PO placebo day 3, IV placebo + PO placebo day 4, and IV placebo + PO Ursodiol 15mg/kg day 5 arm 2: Participants randomized to consume a high fat (72% fat, 8% protein, 20% carbohydrate) meal day 1, then a 100% carbohydrate meal day 2, IV sincalide 0.04mcg/kg + PO placebo day 3, IV placebo + PO placebo day 4, then IV placebo + PO Ursodiol 15mg/kg day 5 arm 3: Participants randomized to consume a 100% carbohydrate meal day 1, then a high fat (72% fat, 8% protein, 20% carbohydrate) meal day 2, IV placebo + PO placebo day 3, IV sincalide 0.04mcg/kg + PO placebo day 4, then IV placebo + PO Ursodiol 15mg/kg day 5 arm 4: Participants randomized to consume a high fat (72% fat, 8% protein, 20% carbohydrate) meal day 1, then a 100% carbohydrate meal day 2, IV placebo + PO placebo day 3, IV sincalide 0.04mcg/kg + PO placebo day 4, the IV placebo + PO Ursodiol 15mg/kg day 5

Interventions: intervention 1: Sincalide intervention 2: Ursodiol intervention 3: Fat Meal intervention 4: Carb meal intervention 5: Placebo
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: 600 calorie meal containing 72% fat, 8% protein, and 20% carbohydrate intervention 4: 600 calorie meal containing 100% carbohydrate intervention 5: IV and/or oral placebo

Protocol document:
Prot_SAP_000.pdf: 
 
1
Clinical Research Protocol 
National Institute of Diabetes, Digestive and Kidney Diseases 
PROTOCOL NUMBER: 08-DK-0165 
PROTOCOL VERSION: 2017_0714 
TITLE: Thyroid hormones homeostasis and energy metabolism changes during 
stimulation of endogenously secreted bile acids (BAs) 
SHORT TITLE: Thyroid, energy metabolism, and thermoregulation  
 
IDENTIFYING WORDS: Bile acids, cholecystokinin, sincalide, thyroid hormones, 
deiodinase, energy expenditure, respiratory quotient (RQ), postprandial thermogenesis 
 
TYPE OF PROTOCOL:  Natural history 
 
START DATE: 03/01/2008   
 
END DATE: July 31, 2018 
 
PRINCIPAL INVESTIGATOR:  
Kong Y. Chen, Ph.D., M.S.C.I 
Clinical Endocrinology Branch (CEB) 
National Institutes of Diabetes, Digestive and Kidney Diseases (NIDDK) 
10 Center Dr. MSC 1613 
BLDG 10, CRC, RM 6-3940 
Phone 301-451-1636 
Email: chenkong@niddk.nih.gov 
 
LEAD ASSOCIATE INVESTIGATOR 
Francesco S. Celi, M.D., MHSc. 
Virginia Commonwealth University 
1101 East Marshall Street  
PO Box 980111  
Sanger Hall, Room 7-007  
Richmond VA 23298-0111  
Phone: 804-828-9696  
Email:  fsceli@vcu.edu 
 
ASSOCIATE INVESTIGATORS:  
Theo Heller, M.D. 
Liver Disease Branch (LDB) 
National Institutes of Diabetes, Digestive and Kidney Diseases (NIDDK) 
Bldg. 10, 9B16 
10 Center Dr. MSC 1800 
Phone 301- 402-7147 
Email: theoh@intra.niddk.nih.gov 
 


 
2
Robert D Shamburek, M.D.  
 
 
 
 
 
Cardiology Branch  
 
 
 
 
 
National Heart, Lung, and Blood Institute  
 
  
(NHLBI)  
 
 
 
 
 
 
Building 10 - Magnuson CC, Room 7N105   
 
10 Center Dr  MSC 1666  
 
 
 
 
Phone: 301 496 3460  
 
 
 
 
Email : bs90j@nih.gov 
 
Alan T. Remaley, MD CC/DLM 
10 Center Dr. MSC 1508 
Bldg.10 Rm. 2C433 
Phone: 301-402-9796 
Email: aremaley1@mail.nih.gov 
 
Frank Pucino, PharmD, MPH 
Clinical Investigator 
DHHS/NIH/NIAMS & NIDDK 
10 Center Drive, 10/CRC 5-2340 NE 
Bethesda, MD  20892-1102 
Phone: (301) 451-5185 
Cell: (240) 446-1961 
Email: frank.pucino@fda.hhs.gov 
 
Joyce Linderman, R.N. 
CEB, NIDDK 
Building 10, CRC, Rm 6-3940 
10 Center Drive, MSC 1613 
Phone: 301-451-7006 
Email: jl626m@nih.gov  
 
 
Sheila Smith, RN 
CEB, NIDDK 
Building 10, CRC, Rm 6-3940 
10 Center Drive, MSC 1613 
Phone: 301-451-1180 
Email: ss1019x@nih.gov 
  
 
 
RESEARCH CONTACT: 
 
Joyce Linderman, R.N. 
CEB, NIDDK 
Building 10, CRC, Rm 6-3940 


 
3
10 Center Drive, MSC 1613 
Phone: 301-451-7006 
Email: jl626m@nih.gov 
 
ABOUT THE PROTOCOL: 
PROJECT DOES NOT USE IONIZING RADIATION. 
PROJECT DOES NOT INVOLVE AN IND/IDE. 
PROJECT DOES NOT USE “DURABLE POWER OF ATTORNEY”. 
STUDY LOCATION IS THE CLINICAL CENTER, NIH. 
THIS IS NOT A MULTI-INSTITUTIONAL PROJECT. 
 


 
4
Précis 
 
Postprandial thermogenesis, or “thermic effect of food” are terms that describe the 
increase in utilization of energy by the human body following a meal. The mechanisms 
involved in this process are believed to differ according to the type of food consumed, 
whether fat, protein or carbohydrate.  
The bile acids (BAs), unique substances secreted by the gall bladder into the gut after a 
meal, play an important role in the absorption of fat and the management of cholesterol 
stores in the body. Recent studies suggest that BAs may also serve as regulators of energy 
expenditure (consumption) in the cells of our body by increasing the production of T3, an 
active form of thyroid hormone. T3 in turn is believed to increase the efficiency with 
which our bodies burn calories thereby generating heat.  Although this process has been 
shown to be effective in rodents who demonstrated weight loss after treatment, the role of 
BAs in humans is poorly understood. Thus we do not know whether endogenous 
(produced by the body) or exogenous (taken as medication) BAs play a significant role in 
the maintenance of body weight. We hypothesize that, similarly to rodents, humans will 
respond to BAs by increasing energy expenditure via the production of the active form of 
thyroid hormone. 
 
This randomized, cross-over study will look at changes in thyroid hormones and energy 
consumption in response to stimuli of endogenous BA secretion including dietary 
content, and to the intake of pharmacological doses of bile acids. 
 
Following a two-day period of equilibration diet, 30 healthy volunteers will be randomly 
assigned to receive either a high-fat or high-carbohydrate isocaloric meal followed by a 
6-hour metabolic chamber stay; the next day they will be crossed-over to the alternate 
intervention.  During the following three days, the study subjects will again be 
randomized to receive either an intravenous injection of sincalide (the C-terminal 
octapeptide fragment of cholecystokinin) 0.04 mcg/kg or placebo and P.O. placebo, or 
I.V. placebo and 15 mg/kg of BA (ursodiol) with similar metabolic chamber stays and 
cross-over design. 
The following parameters will be recorded and compared to placebo: 
 
 
Energy expenditure 
Substrate utilization 
Spontaneous movements 
Skin and core temperature 
Serial changes in circulating thyroid hormones 
Serial changes in bile acid serum concentrations  
 
The data gathered from this study will provide greater insight into the physiological and 
molecular mechanism(s) regulating the relation between endogenous bile acid secretion 
and energy metabolism in response to meals, as well as the role of BAs per se on energy 
metabolism. 


 
5
Introduction 
 
The overall energy balance is maintained by multiple redundant mechanisms 
regulating both energy intake and expenditure (EE) (1, 2). While a considerable amount 
of experimental and clinical data on the mechanisms regulating food intake is currently 
available, comparatively little is known about the regulation of postprandial 
thermogenesis (PPT). Classical studies by Rubner (3) and Lusk (4) have revealed the 
unique role of protein in PPT and termed it “specific dynamic action”. However, the roles 
of fat and carbohydrate, the major sources of energy intake, on PPT are less clear. 
Recently, Blaak et al. (5) have reported that the level of increase in 3-hr postprandial EE 
on a high-fat (95%) meal was lower in obese versus individuals. On the other hand, it has 
been shown that the metabolism of 14C labeled dietary oleic acid to14CO2 began after 6 
hours and end around 15 hours. Thus, the cause for this acute rise in PPT (<6 hours) 
cannot be attributed directly to the oxidation per se of the dietary fat.  
BAs are steroid acids found predominantly in the bile of mammals. In humans, 
the most important BAs are cholic acid, chenodeoxycholic acid, and their conjugates with 
taurine and glycine (glycocholate and taurocholate). These molecules are derived from 
cholesterol and synthesized endogenously by the liver. Storage of excess bile acid takes 
place within the gall bladder from where it is available for secretion upon stimulation by 
such factors as cholecystokinin (CCK) and high-fat diet. CCK, a gut-peptide hormone, 
naturally secreted by the duodenum in response to fat and protein rich meals causes 
increased production of hepatic bile, stimulation of gallbladder contraction and the 
relaxation of the Sphincter of Oddi, resulting in the delivery of bile into the duodenum. 
CCK and high-fat meals induce, together with an increase in the secretion of bile in the 
duodenum, a rapid rise in the plasma concentration of BAs(6). 
BAs have long been known to play a central role in lipid, fat soluble vitamin 
absorption and cholesterol catabolism, however in recent years an important role for BAs 
as signaling molecules has emerged. BA binding to FXR nuclear receptor can reduce 
expression of 7-alpha hydroxlase a rate-limiting enzyme in the synthesis of BAs in 
hepatic cells. Interestingly, BAs also act through an alternate pathway by binding a cell 
surface receptor TGR5. TGR5 is a G protein coupled receptor (GPCR), which upon 
binding with its ligand, leads to an increase in intracellular cAMP production. Recent 
evidence indicates that the cAMP-driven Type-2 Deiodinase gene (D2) is the naturally 
occurring target of the TGR5 signal pathway, ultimately resulting in an increase in the 
intracellular concentration of T3(7, 8) the active form of thyroid hormone. 
This phenomenon has been demonstrated in BAT of mice and human skeletal 
muscle cells, tissues that express both TGR5 and D2 (8). In rodents fed a high fat diet,  
cholic acid prevented the development of diet-induced obesity by increasing energy 
expenditure (EE) and oxygen consumption in BAT through increased D2-mediated T4 to 
T3 conversion (8-11) This effect was not observed in D2 knockout mice or those rodents 
fed a regular diet. These data can thus be interpreted as an endocrine action of the bile 
acids whose postprandial serum levels increase would lead to an increase in D2 
transcription and activity ultimately generating an increase in thermogenesis, thus 
responding in a homeostatic fashion to the caloric load. It is postulated that elevated 
intracellular cAMP and T3 concentrations induce the expression of uncoupling proteins 
(UCPs)(12). The UCPs, by reducing the “efficiency” of mitochondrial oxidative 


 
6
phosphorylation, generate heat and thereby increase EE. In rodents this is achieved by the 
activation of the UCP-1 in the brown adipose tissue (BAT). Indeed, the UCP-1 gene is a 
thyroid-hormone dependent gene, and mice devoid of D2 are unable to produce non-
shivering thermogenesis in response to cold exposure.  
In humans, the pattern of expression of D2 is strikingly different from the 
rodent(13, 14). Specifically, while adult individuals have no “anatomically defined” 
BAT, D2 and UCP-3 have been demonstrated in skeletal muscle, raising the possibility 
that this tissue, which is approximately 40-80% of the individual’s weight, could 
represent a “physiological equivalent” of BAT(10, 15). UCP-3 induction in skeletal 
muscle, similar to UCP-1 in rodents, may mediate an increase in EE after BA treatment 
thereby promoting weight loss and insulin sensitivity.  Indirect evidence suggests that the 
D2-mediated T4 to T3 conversion plays a major role in the production of the circulating 
T3(16). Interestingly, two common genetic variants of the human D2 gene, Thr92Ala and 
-258 A/G SNP, have been associated both in cross sectional and in in vitro studies with 
changes in the enzymatic activity (17-20).  
  
At the present, the role of BAs in the regulation of metabolism and energy 
expenditure in human BAT or “BAT-physiological equivalent” is unclear. In vitro 
evidence from cultured human skeletal muscle cells suggests that BAs increase the 
metabolic rate in this tissue. If this is true, one could hypothesize that the manipulation of 
circulating bile acid levels, by pharmacologic or dietary means, could result in an overall 
increase in EE, ultimately facilitating weight loss. Data available in the gastroenterology 
literature clearly demonstrate that either a high-fat meal or a single injection of CCK is 
able to generate respectively a 1.5 to 3-fold and 2-fold rises in circulating BAs (6, 21, 22) 
without any significant residual effect to the following day(23). Interestingly, the chronic 
use of bile acid salts for the treatment of primary biliary cirrhosis resulted in a mild 
increase in body weight(24). This finding was attributed to an overall improvement of the 
general (nutritional?) conditions of the patients (Dr K. Lindor, Mayo Clinic, personal 
communication). No data is currently available relative to the synchronous evaluation of 
energy expenditure and endocrine metabolism during manipulation of endogenous 
secretion or circulating levels of BAs. 
 
The recently opened Metabolic Unit, a state-of-the-art metabolic phenotype 
characterization laboratory, has the capability, through the temperature-controlled and 
fast response metabolic chambers, to analyze changes in the human physiology in 
controlled environmental conditions. This hypothesis-driven natural history 
protocol/physiology is thus aimed, by taking full advantage of the capabilities of the 
Metabolic Unit, to the detailed characterization of the physiological response to the 
stimulation of endogenous BA secretion and to characterize the role of BAs in the 
postprandial thermogenesis.  
 
Study Hypotheses 
 
1) If BAs have an endocrine/paracrine action on BAT-equivalent tissue in humans, the 
acute pharmacological stimulation of the endogenous BAs secretion by CCK and/or the 
oral administration of BAs will result in an increase in thermogenesis similar to the one 


 
7
observed in the postprandial period of a high-fat meal, and greater than the one observed 
in a high-carbohydrate one. 
 
2) If the raise in circulating BAs is coincidental but not causative of the increase in 
thermogenesis observed in the postprandial period, we do not expect to observe any 
change in EE after an oral dose of BAs. Consequently, if the increase in thermogenesis 
observed in the postprandial period is secondary to endocrine effects of CCK, but not to 
BAs per se, we will observe an increase in EE after CCK infusion but not after an oral 
dose of BAs. 
 
3) Conversely, if the postprandial thermogenesis is secondary to calorie intake, 
irrespective of macronutrient composition, while no thermogenic effect will be observed 
after acute administration of BAs (or the pharmacological stimulation of the endogenous 
BAs secretion), a comparable increase in thermogenesis will be observed after a high 
carbohydrate or a high-fat meal. 
 
4) If the postprandial thermogenesis is dependent to the secondary macronutrient 
composition, a differential increase in the thermogenesis will be observed after a high 
carbohydrate as compared to a high-fat meal.  
 
Aim of the study: 
 
1) To characterize the metabolic and hormonal responses to changes in endogenous BAs 
secretion following CCK stimulation or pharmacological administration of BAs. 
 
2) To characterize the role of BAs and macronutrient composition in postprandial 
thermogenesis. 
 
Secondary aims 
 
1) To characterize the role of genetic variants of the type-2 deiodinase gene (Thr92Ala 
and -258 A/G) in the modulation of the hormonal and metabolic response to changes in 
circulating BAs. 
 
2) To analyze the changes in glucose metabolism and substrate utilization in response to 
changes in circulating BAs. 
 
 
Study Protocol 
 
Subjects and Recruitment 
 
Subjects will be healthy non-obese (BMI<27) male and female adult volunteers.   
 
 
 


 
8
Study design and methods 
 
This natural history research protocol is designed as a double blind-cross over study with 
both pharmacological and nutritional interventions. During a seven-day stay at the NIH, 
volunteers will undergo five 6-hour clinical and experimental observations in the 
metabolic chamber. The timing of the experiments have been based on the observations 
of Hoffman and colleagues who have shown that the peak of BA’s occurs at ~2 hours 
postprandially in a physiological pharmacokinetic model in man (25) and of Foberg et al. 
who showed that CCK induced a rise in serum bile acids (3α-HBA) peaked at ~30 
minutes and returned to baseline within  120 min (6). The overall outline of the study is 
reported in Figure 1. Briefly, eligible volunteers will be invited for a seven-day stay at the 
Metabolic Unit. After a two-day isocaloric equilibration diet, study volunteers will be 
randomly assigned to receive either a very low or high-fat isocaloric standardized liquid 
meal during a 6-hour stay in the metabolic chamber. After an 18-hour resting period the 
volunteers will then be crossed-over to the second type of meal. After an 18-hour resting 
period the volunteers will again be randomly assigned to receive an IV injection of 
sincalide (Kinevac, a sterile lyophilized powder of the C-terminal octapeptide fragment of 
cholecystokinin) 0.04 mcg/kg or placebo during a 6-hour stay in the metabolic chamber. 
After an 18-hour post infusion resting period, the volunteers will be crossed-over to the 
alternate study arm; during these two interventions the volunteers will also receive a P.O. 
placebo. The next day, after an 18-hour post infusion resting period from the previous 
metabolic chamber stay, the volunteers will receive an IV infusion of placebo and 15 
mg/kg of ursodiol (as 300 mg capsules rounded up to the nearest 300 mg), and will 
undergo the same recording. The volunteers will be blinded relative to the composition of 
the IV and PO compounds. This study design is necessary to avoid carry-over effects of 
the PO BAs. 
 


 
9
 
 
During each 6-hour stay in the metabolic chamber we will record the following 
parameters and will perform these procedures (Fig. 2, A: High fat/High carbohydrate 
meal, B: Sincalide/IV Placebo-P.O. Placebo, C: IV Placebo-ursodiol): 


 
10
 
1) Real-time energy expenditure, RQ 
2) Spontaneous movements 
4) Real-time core and skin temperature 
5) Frequent blood samples for the following parameters: 
 
Free fatty acids 
 
TSH 
 
T4 
 
T3 
 
 
Free T4 
 
Free T3 
 
rT3 
 
 
Glucose 
 
Insulin 
 
Bile acid levels 
 
Ghrelin 
 
CCK 
 
GLP-1 
 
-hydroxybutirrate 
 
Interleukin-6, 
 
Adiponectin. 
 
 
 


 
11
Inclusion Criteria: 
• Age ≥18 years, male or female. 
• Written informed consent. 
 
Exclusion Criteria: 
 
• Hypo- or hyperthyroidism (history or serum TSH > 5.0 or < 0.4 miU/L) 
• Blood pressure greater than 140/90 mmHg (26) or receiving antihypertensive 
therapy 
• History of cardiovascular disease 
• BMI ≤ 20 or ≥ 27 Kg/m2. 
• Diabetes mellitus (fasting serum glucose ≥ 126 mg/dL). 
• Hyperlipidemia (serum total cholesterol ≥ 240 mg/dL, triglycerides ≥ 220 mg/dL, 
and/or use of antilipemic therapy). 
• Liver disease or ALT serum concentrations greater than 1.5 times the upper 
laboratory reference limit. 
• Hyperbilirubinemia (serum total bilirubin > 1.5 mg/dL) 
• Renal insufficiency or estimated creatinine clearance ≤ 50 mL/min (MDRD 
equation). 
• Anemia (Hemoglobin concentration < 11.1 g/dL females , and 12.7 g/dL  males) 
• History of cholecystectomy or cholelithiasis (by ultrasound at screening). 
• History of malabsorption, or food allergies/intolerances that would preclude 
participant from consuming foods required for study 
• Claustrophobia. 
• History of illicit drug or alcohol abuse within the last 5 years; current use of illicit 
drugs (by history) or alcohol (CAGE > 3). 
• Psychiatric conditions or behavior that would be incompatible with safe and 
successful participation in this study 
• Current use of medications/dietary supplements/alternative therapies known to 
alter thyroid function, energy expenditure or bile acid secretion. 
• History of weight loss or weight gain of >3% body weight over the past 2 months 
(self-reported) 
• Pregnancy/breastfeeding/hormonal contraceptive use and childbirth within the last 
6 months. 
• Perimenopausal (as self-described within two years from onset of amenorrhea or 
current complaints of hot flashes) 
• Current smoker 
 
All subjects will be fully informed of the aim, nature, and risks of the study prior to 
giving written informed consent. 
 
 
 
 
 


 
12
 
 
Methods:  
 
Screening visit 
Subjects will be screened and counseled at the initial screening visit. After obtaining 
informed consent, the study volunteers will undergo a history and physical examination, 
and the following screening tests will be performed, including: TSH, glucose, creatinine, 
AST, ALT, CBC, cholesterol, EKG and gall bladder ultrasound. Subjects are required to 
fast for at least 12-hour prior to the laboratory testing, but will be allowed to drink water 
during the fasting period. During the screening visit, the study participants will be 
interviewed by a dietitian in order assess the caloric intake and food preferences of each 
study participant. 
 
Hospital stay 
Study volunteers will be housed in the Metabolic Unit of the NIH Clinical Center. Non-
menopausal female volunteers will be admitted during the early follicular phase (day 2-5) 
of the menstrual cycle and a pregnancy test will be performed at admission. During the 
hospital stay, the volunteers will be asked not to perform strenuous physical activity and 
to abstain from tobacco use. After two days of equilibration diet the subjects will be 
randomly assigned to receive low and high fat meal during the metabolic chamber stay. 
The next day the volunteers will receive the alternate meal. The following day the 
volunteers will be again randomized to sincalide or placebo and P.O. placebo; the next 
day volunteers will receive the alternate treatment. In the last day of hospital stay the 
volunteers will again receive IV placebo and 15 mg/kg of ursodiol. 
 
Metabolic Diet 
Throughout the stay at the Metabolic Unit study volunteers will receive an isocaloric, 
caffeine-free diet with the following macronutrient distribution: 50% carbohydrate, 20% 
protein, 30% fat.  A research dietitian will meet with participants during the screening 
visit to plan the isocaloric equilibration diet and to assess food allergies and intolerances.  
Approximate total energy needs for weight maintenance will be calculated using the 
Mifflin-St.Jeor equation (27).   During two of the metabolic chamber stays study 
volunteers will be randomized to receive an isocaloric high fat or fat free meal.  After an 
18 hour resting period the subjects will be crossed over to the second type of meal.  Both 
of the meals will consist of a shake with approximately 600 calories.  The high fat meal 
will contain 50 grams of fat and will have an approximate macronutrient distribution of 
72% fat, 8% protein, and 20% carbohydrate.  The fat free meal will contain 0 grams of fat 
and will be 100% carbohydrate.  The shakes to be consumed in ≤15 min, will be similar 
in volume and will be served at room temperature. 
 
 
Body composition 
A DEXA scan and an air-displacement plethysmography body composition analysis 
(BodPod) will be performed at either day 1 or 2 of the hospital stay. 
 
 


 
13
Metabolic chamber 
Study volunteers will enter the metabolic chamber at 08:00 AM after a 10-hour fast, 
voiding, and ingestion of the core temperature probe. Superficial skin temperature probes 
will also be placed (see below). Study volunteers will wear a standard suit (hospital 
scrub) and will be allowed to perform regular physical activity while in the metabolic 
chamber. 
 
The respiratory chamber is a specially constructed room to assess the metabolism 
of subjects for a period of 6 hours in this study under constant thermoneutral condition 
(73.5±0.5° F).  Designed as a walk-in “pull” calorimeter, it is an open circuit unit that 
draws conditioned room air into the chamber at the same flow rate as it is extracted into 
the gas analysis system.(28)  The room is equipped with toilet and sink with privacy 
screen, treadmill, bed, desk, and computer with access to television and other forms of 
entertainment.  Physical activity level is measured continuously through a wall mounted 
monitoring device (microwave sensor).  Food and fresh water is passed through an air-
lock drawer system. Using this rapid response chamber, we will first measure the resting 
EE after the patient enters the room. While still fasted, the subject will lie in supine 
posture and with minimum movement for 30 minutes. After each meal, the subject will 
be instructed to remain sedentary for 2 hours where the postprandial thermogenesis (EE 
above the resting level) will be determined. The energy cost of spontaneous physical 
activity (29) will also be assessed. The respiratory quotient (RQ), as determined by the 
ratio of carbon dioxide excretion and oxygen consumption, during resting, post-prandial, 
and overall 6-hour periods will be used to estimate whole-body carbohydrate and fat 
oxidation rates. 
 
 
The following parameters will be recorded:  
 
Spontaneous movements 
Spontaneous physical movements will be quantified with minute-to-minute resolutions 
during the study periods using small, portable pager-type and watch accelerometers at the 
subjects’ hip and wrist (30).   
 
Real-time core and skin temperature 
The VitalSense® monitor (Mini Mitter/Respironics,  Bend OR) is used to receive 
transmissions from multiple miniature, wireless, temperature sensors. Core temperature is 
sensed by the ingestible Jonah™ capsule. Dermal temperatures are recorded from 
hypoallergenic, non-irritating, adhesive dermal patches. Both sensor types are disposable, 
but designed for multi-day use under demanding physical and environmental conditions. 
Both types of sensors use low-power radio frequency transmissions to communicate with 
the Monitor.  The core temperature capsules transmit at various frequencies, therefore, it 
is possible to track more than one capsule at a time. 
 
Blood sampling, urine collection 
All blood samples will be drawn under sterile technique through an indwelling catheter 
using the Vacutainer® system (Becton Dickson and Co.). Serum and plasma aliquots will 
be stored at -80 C until analysis. Blood samples will be drawn according to the 


 
14
following scheme during each metabolic chamber stay: -15’,0’,15’,30’, 45’, 60’, 75’, 90’, 
120’, 150’, 180’, 240’, 300’,360’. 
The following laboratory parameters will be recorded: T4, T3, free T4, free T3, TSH, 
glucose, insulin, BAs, Ghrelin, CCK, GLP-1, and free fatty acids. 
 
 
 
Genetic testing 
DNA samples will be obtained from leukocytes obtained during the screening visit. 
Samples will be analyzed for the Thr92Ala and -258 A/G polymorphisms, and for 
subsequent screening of known mutations of genes involved in the energy, carbohydrate, 
thyroid hormone and glucose metabolism, as well as scanning for novel mutations in 
genes involved in these same metabolic pathways. Since these data do not currently have 
clinical relevance, study volunteers will not be notified of the results. 
 
Laboratory testing  
Screening tests (Chem 20, lipid panel, CBC with differential, TSH, HCG) will be 
performed in the Department of Laboratory Medicine (DLM). Genomic DNA will be 
isolated from peripheral mononuclear cells using the QIAmp® system (QIAGEN) in Dr. 
Celi’s laboratory. The following tests will be performed by the Department of Laboratory 
Medicine (DLM): Chem 20, insulin, cholesterol, HDL, triglycerides, free fatty acids, 
TSH, total and free T3, and total and free T4. Aliquots of serum and plasma will be also 
collected and stored at -80 C for further analysis. GLP-1, CCK, and Ghrelin will be 
measured by immunometric assay in the clinical research core laboratory of NIDDK. 
Serum BAs (Cholic acid, Deoxycholic acid, Chenodeoxycholic acid, Total bile acids) 
will be measured by radio-immunoassay technique (Mayo Clinic). 
 
Radiology studies 
Right upper quadrant ultrasound to screen for the presence of gallstones will be 
performed at the screening visit. DEXA scan for body composition will be performed 
during hospital day #1 or #2 using the Metabolic Unit GE iDXA.  
 
Statistical analysis 
 
The primary endpoint of this mechanistic study is the analysis in changes in energy 
expenditure in response to changes (pharmacological or diet-induced) in serum bile acids 
concentration. The data will be analyzed using Analysis of Variance (ANOVA) and, 
should the data suggest an interaction, we will explore statistical models including delta 
in bile acid concentration, thyroid hormone concentration, and macronutrient diet (as a 
categorical data). Power analysis: Although this is a pilot study, we use previously 
published data comparing the effect on PPT (31) to help power the study, The PPT was 
7.6-10.1% of the total energy intake, with a standard deviation of 2.6%. If we detect a 
desired difference of 2.0% in PPT with CCK or high fat diets, 28 study subjects will be 
needed to provide an 80% statistical power ( error of 0.05, using a two-tailed 
independent t-test design). We plan to enroll 30 volunteers considering the possibility of 
missing values. 


 
15
 
Research use, storage, and disposition of human subject’s samples and data 
 
Serum, plasma, and nucleic acid samples will be coded and stored in a -80C refrigerator 
located in the PI’s laboratory. Tissue samples will be coded and stored in a liquid 
nitrogen tank located in the PI’s laboratory. A registry of the samples location will be 
kept in the PI’s office. This will allow the PI and the AIs to analyze research data in 
relation to clinical data stored in the Medical Record Department. Data and samples will 
be retained for the foreseeable future in order to allow secondary analyses after the 
completion of the protocol. Such analyses include association studies for known 
mutations of genes involved in the energy, carbohydrate, thyroid hormone and glucose 
metabolism, as well as gene scanning for novel mutations. Serum, plasma and nucleic 
acids samples will be similarly utilized for future association studies of known and novel 
markers of energy, carbohydrate, thyroid hormone and glucose metabolism. The PI will 
inform the IRB in case of loss of data/samples secondary to intrusion in the laboratory or 
database. 
 
Possible Risks and Hazards: 
 
This study, aimed to analyze the role of BAs in postprandial thermogenesis, is not a 
therapeutic trial. Research-related risks in this study include those associated with study 
procedures, namely blood drawing. We do not expect serious study medication-related 
risks except nausea or mild and transient diarrhea (see below) due to the single injection 
of sincalide or to a single dose of ursodiol, nor do we expect any significant risk 
connected to the respiratory chamber stay.  
 
Metabolic Chamber. 
Besides inconveniences that can reasonably be expected as a result of spending 6 hours in 
the live-in room calorimeter, the serious risk to subjects' health is minimum.  
 
Blood drawing.  
The placement of intravenous needles may cause transient pain, and occasional infection 
or bruising at the insertion site. Study subjects will undergo 5 venipunctures (14 blood 
draws during each metabolic chamber stay) during the study. Each blood drawing will be 
for 5 mL of blood. However, in compliance with NIH guidelines, no more than 6 mL/Kg 
will be drawn from any study participants during a six-week period. 
 
Core-body and surface temperature measurements.  
A small temperature "pill" and wireless surface patches (VitalSense, Mini Mitter, Bend 
OR) will be used to measure the diurnal variation of body temperature, and a small 
receiver will need to remain close to the body.  The "temp pill" is a capsule (8.7 mm x 23 
mm) which is swallowed with water and will be stay in the digestive tract for 2-3 days 
until being eliminated in the feces.  Patients are advised not to undergo MRI procedure 
until the capsule is passed. No other known side-effect has been reported. 
 
Frequent blood-pressure monitoring.  


 
16
An ambulatory blood pressure monitor (Oscar 2, SunTech Medical, Morrisville NC), 
using a cuff on the upper arm, will be programmed to measure BP very 15 minutes. 
During the measurement, temporary pressure/restriction to the arm is typical. Minor pain 
may be experienced if the cuff is too tight. Adjustment of the cuff is allowed. No other 
known side-effect has been reported. 
 
 
DEXA Scan.   
The use of DEXA scan apparatus may cause some minimal discomfort in claustrophobic 
subjects and may cause some minimal back pain in a small minority of the individuals. 
 
Radiation Safety.   
The radiation exposure of a whole-body DEXA Scan is less than 1 mrem.  A typical 
radiation dosage from a chest X-ray is 20 mrem.  A normal individual is exposed to 300 
mrem/year from natural sources. 
 
 
 
Study drugs: 
 
Sincalide (Kinevac) 
Reactions to sincailde are generally mild and of short duration. Common adverse effects 
(reported in 20% of patients) associated with sincalide include abdominal pain, cramps, 
and nausea. Less frequent side effects, such as dizziness, diaphoresis, diarrhea, dyspnea, 
fecal urgency, flushing, headache, hyper-/hypotension, rash, sneezing, numbness, and/or 
vomiting occur in < 2% of patients. 
 
 Ursodeoxycholic Acid  
Possible side effects occurring in more than 1% of patients receiving ursodiol for 
cholecystitis or primary biliary cirrhosis have included: headache (up to 25%), dizziness 
(up to 17%), constipation (up to 26%), rash (<1%-3%), hair loss (<1% to 5%), diarrhea 
(1%), nausea, vomiting (felt to be related to cholecystitis), musculoskeletal pain (6-8%), 
cough or respiratory infections (5-15%), leukopenia (3%), and allergy (5%). The 
incidence of many of these side effects was similar to those reported in the placebo 
group, and, according to product labeling, doses of ursodiol in the range of 16-20 
mg/kg/day have been tolerated in some patients for 6-37 months without symptoms. 
 
 
Acetaminophen (Tylenol) 
This analgesic will be prescribed for headache and minor pain. For the dose and duration 
of use in this study, serious adverse effects would not be expected. 
 
Event Characterization and Reporting to the IRB and Clinical Director (CD) 
 
 
Adverse events, protocol deviations, unanticipated problems (UP), Unanticipated Adverse 
Device Effects (UADEs), serious adverse events, sponsor and serious, are defined as 


 
17
described in NIH HRPP SOP 16 (“Reporting Requirements for Unanticipated Problems, 
Adverse Events and Protocol Deviations.”). All adverse events occurring during the 
study, including those observed by or reported to the research team, will be recorded. 
Serious unanticipated problems and serious protocol deviations will be reported to the 
IRB and CD as soon as possible but not more than 7 days after the PI first learns of the 
event. Not serious unanticipated problems will be reported to the IRB and CD as soon as 
possible but not more than 14 days after the PI first learns of the event. Not serious 
protocol deviations will be reported to the IRB as soon as possible but not more than 14 
days after the PI first learns of the event. 
 
Deaths are not expected to occur since no subjects are being recruited. In the unlikely event 
that a death is reported to the Investigator, it will be reported to the Clinical Director and 
IRB within 7 days after learning of the event. 
 
Non-Serious Protocol Deviations 
 
Non-serious protocol deviations will only be reported to the IRB (within 14 days after the 
PI first learns of the event) if they represent a departure from NIH policies for the 
conduct of human subjects research, adversely affect the health care of the subject(s) or 
compromise the interpretation or integrity of the research.  Non-serious protocol 
deviations that result from technical issues associated with sampling that does not impact 
the health of the subject or the interpretation of the study data will not be reported. 
 
Adverse Events 
 
The PI is responsible for summarizing all serious adverse events and adverse events at 
least possibly related to the research procedure and interventions at the time of 
Continuing Review. 
 
Data and Safety Monitoring Plan 
 
Due to the modest risk of the study procedures, no DSMB will be instituted to evaluate 
adverse events. For this protocol the PI will serve as study monitor. 
 
Study procedures will be subject to audits and/or monitoring visits to ensure compliance 
with the protocol and applicable regulatory requirements consistent with the NIDDK 
quality assurance program plan.  Audit and/or monitoring visit results will be reported to 
the Principal Investigator for further reporting as appropriate.   Study documents and 
pertinent hospital or clinical records will be reviewed to verify that the conduct of the 
study is consistent with the protocol plan. 
 
 
Recruitment of Women, children and minority individuals 
 
We will actively encourage the participation of women and minorities. On the other hand, 
the study design precludes the recruitment of children, since the differences in the 


 
18
biomarkers between adults and children would impede the interpretation of the data 
collected. Moreover, this is not a therapeutic trial and children will not be prevented from 
receiving potentially beneficial therapy by the exclusion from this study.  
 
Benefits 
 
Study subjects will receive no direct benefit from participation in this study. However, a 
thorough medical examination, and a series of diagnostic tests will be provided as part of 
the evaluation. Abnormal values will be discussed with the study volunteers and 
forwarded to their primary care physicians.  
 
Remuneration 
 
Patients will receive payments for time and discomfort connected with the 
visits/procedures according to the following scheme: 
 
1. Daily compensation for impatient stay/venipunctures 
$250 
2. Inconvenience for the study medications administration 
$50 
3. Compensation for metabolic chamber stay ($100 each) 
$500 
4. Compensation for completion of the study 
 
 
$100 
 
Maximum compensation 
 
 
 
 
 
$900 
 
In case of premature termination of the study the volunteers will be remunerated only for 
the actual procedures, and the bonus compensation for the completion of the study, as 
well as the study medications administration will be forgone. 
 
 
Confidentiality 
 
Study data will be kept in locked files, and subjects will be identified by codes.  
 
Genetic testing 
 
DNA samples will be obtained from leukocytes obtained during the screening visit. For 
subsequent screening of known mutations of genes involved in the energy, carbohydrate, 
thyroid hormone and glucose metabolism, as well as scanning for novel mutations in 
genes involved in the energy, carbohydrate, thyroid hormone and glucose metabolism. 
Since these data do not currently have clinical relevance, study volunteers will not be 
notified of the results. 
 
 
Consent process and documents 
 
Written informed consent will be obtained at the time of the screening visit and 
confirmed before the procedure.  


 
19
REFERENCES 
 
1. 
Hill JO 2006 Understanding and addressing the epidemic of obesity: an energy 
balance perspective. Endocr Rev 27:750-61 
2. 
Rosen ED, Spiegelman BM 2006 Adipocytes as regulators of energy balance and 
glucose homeostasis. Nature 444:847-53 
3. 
Rubner M 1902 Die Gesetze des Energieverbrauchs bei der Ernährung. Franz 
Deuticke, Lepizig 
4. 
Lusk G 1928 The science of nutrition, 4th ed ed. WB Saunders, Philadelphia 
5. 
Blaak EE, Hul G, Verdich C, Stich V, Martinez JA, Petersen M, Feskens EF, 
Patel K, Oppert JM, Barbe P, Toubro S, Polak J, Anderson I, Astrup A, 
Macdonald I, Langin D, Sorensen T, Saris WH 2007 Impaired fat-induced 
thermogenesis in obese subjects: the NUGENOB study. Obesity (Silver Spring) 
15:653-63 
6. 
Foberg U, Fryden A, Kagedal B, Pettersson L, Tobiasson P 1987 Comparison 
of three bile acid provocation tests. Intravenous cholecystokinin, a standard test 
meal, and an oral bile acid load in healthy subjects. Scand J Gastroenterol 22:606-
8 
7. 
Bianco AC, Silva JE 1988 Cold exposure rapidly induces virtual saturation of 
brown adipose tissue nuclear T3 receptors. Am J Physiol 255:E496-503 
8. 
Watanabe M, Houten SM, Mataki C, Christoffolete MA, Kim BW, Sato H, 
Messaddeq N, Harney JW, Ezaki O, Kodama T, Schoonjans K, Bianco AC, 
Auwerx J 2006 Bile acids induce energy expenditure by promoting intracellular 
thyroid hormone activation. Nature 439:484-9 
9. 
Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 
Biochemistry, cellular and molecular biology, and physiological roles of the 
iodothyronine selenodeiodinases. Endocr Rev 23:38-89 
10. 
Lanni A, Moreno M, Lombardi A, Goglia F 2003 Thyroid hormone and 
uncoupling proteins. FEBS Lett 543:5-10 
11. 
de Jesus LA, Carvalho SD, Ribeiro MO, Schneider M, Kim SW, Harney JW, 
Larsen PR, Bianco AC 2001 The type 2 iodothyronine deiodinase is essential for 
adaptive thermogenesis in brown adipose tissue. J Clin Invest 108:1379-85 
12. 
Silva JE 1995 Thyroid hormone control of thermogenesis and energy balance. 
Thyroid 5:481-92 
13. 
Croteau W, Davey JC, Galton VA, St Germain DL 1996 Cloning of the 
mammalian type II iodothyronine deiodinase. A selenoprotein differentially 
expressed and regulated in human and rat brain and other tissues. J Clin Invest 
98:405-17 
14. 
Salvatore D, Bartha T, Harney JW, Larsen PR 1996 Molecular biological and 
biochemical characterization of the human type 2 selenodeiodinase. 
Endocrinology 137:3308-15 
15. 
Choi CS, Fillmore JJ, Kim JK, Liu ZX, Kim S, Collier EF, Kulkarni A, 
Distefano A, Hwang YJ, Kahn M, Chen Y, Yu C, Moore IK, Reznick RM, 
Higashimori T, Shulman GI 2007 Overexpression of uncoupling protein 3 in 
skeletal muscle protects against fat-induced insulin resistance. J Clin Invest 
117:1995-2003 


 
20
16. 
Maia AL, Kim BW, Huang SA, Harney JW, Larsen PR 2005 Type 2 
iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans. J 
Clin Invest 115:2524-33 
17. 
Mentuccia D, Proietti-Pannunzi L, Tanner K, Bacci V, Pollin TI, Poehlman 
ET, Shuldiner AR, Celi FS 2002 Association between a novel variant of the 
human type 2 deiodinase gene Thr92Ala and insulin resistance: evidence of 
interaction with the Trp64Arg variant of the beta-3-adrenergic receptor. Diabetes 
51:880-3 
18. 
Canani LH, Capp C, Dora JM, Meyer EL, Wagner MS, Harney JW, Larsen 
PR, Gross JL, Bianco AC, Maia AL 2005 The type 2 deiodinase A/G 
(Thr92Ala) polymorphism is associated with decreased enzyme velocity and 
increased insulin resistance in patients with type 2 diabetes mellitus. J Clin 
Endocrinol Metab 90:3472-8 
19. 
Peeters RP, van den Beld AW, Attalki H, Toor H, de Rijke YB, Kuiper GG, 
Lamberts SW, Janssen JA, Uitterlinden AG, Visser TJ 2005 A new 
polymorphism in the type II deiodinase gene is associated with circulating thyroid 
hormone parameters. Am J Physiol Endocrinol Metab 289:E75-81 
20. 
Coppotelli G, Summers A, Chidakel A, Ross JM, Celi FS 2006 Functional 
Characterization of the 258 A/G (D2-ORFa-Gly3Asp) Human Type-2 Deiodinase 
Polymorphism: A Naturally Occurring Variant Increases the Enzymatic Activity 
by Removing a Putative Repressor Site in the 5' UTR of the Gene. Thyroid 
16:625-32 
21. 
Fausa O 1976 Serum bile acid concentration after a test meal. Scand J 
Gastroenterol 11:229-32 
22. 
LaRusso NF, Korman MG, Hoffman NE, Hofmann AF 1974 Dynamics of the 
enterohepatic circulation of bile acids. Postprandial serum concentrations of 
conjugates of cholic acid in health, cholecystectomized patients, and patients with 
bile acid malabsorption. N Engl J Med 291:689-92 
23. 
Kelbaek H, Linnet K, Stimpel H, Glenthoj J, Thomsen HF, Bahnsen M 1984 
Cholecystokinin-stimulated and postprandial serum concentrations of bile acids in 
alcoholic liver cirrhosis. Scand J Gastroenterol 19:655-60 
24. 
Siegel JL, Jorgensen R, Angulo P, Lindor KD 2003 Treatment with 
ursodeoxycholic acid is associated with weight gain in patients with primary 
biliary cirrhosis. J Clin Gastroenterol 37:183-5 
25. 
Hofmann AF, Molino G, Milanese M, Belforte G 1983 Description and 
simulation of a physiological pharmacokinetic model for the metabolism and 
enterohepatic circulation of bile acids in man. Cholic acid in healthy man. J Clin 
Invest 71:1003-22 
26. 
Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, 
Jr., Jones DW, Materson BJ, Oparil S, Wright JT, Jr., Roccella EJ 2003 The 
Seventh Report of the Joint National Committee on Prevention, Detection, 
Evaluation, and Treatment of High Blood Pressure: the JNC 7 report. Jama 
289:2560-72 
27. 
Mifflin MD, St Jeor ST, Hill LA, Scott BJ, Daugherty SA, Koh YO 1990 A 
new predictive equation for resting energy expenditure in healthy individuals. Am 
J Clin Nutr 51:241-7 


 
21
28. 
Nguyen T, de Jonge L, Smith SR, Bray GA 2003 Chamber for indirect 
calorimetry with accurate measurement and time discrimination of metabolic 
plateaus of over 20 min. Med Biol Eng Comput 41:572-8 
29. 
Schutz Y, Ravussin E, Diethelm R, Jequier E 1982 Spontaneous physical 
activity measured by radar in obese and control subject studied in a respiration 
chamber. Int J Obes 6:23-8 
30. 
Chen KY, Bassett DR, Jr. 2005 The technology of accelerometry-based activity 
monitors: current and future. Med Sci Sports Exerc 37:S490-500 
31. 
Lejeune MP, Westerterp KR, Adam TC, Luscombe-Marsh ND, Westerterp-
Plantenga MS 2006 Ghrelin and glucagon-like peptide 1 concentrations, 24-h 
satiety, and energy and substrate metabolism during a high-protein diet and 
measured in a respiration chamber. Am J Clin Nutr 83:89-94