Combined Effects of GLP-1 Analogue and Exercise on Maintenance of Weight Loss and Health After Very-low Calorie Diet
Combined Effects of GLP-1 Analogue and Exercise on Maintenance of Weight Loss and Health After Very-low Calorie Diet
Introduction: The success rate of weight loss maintenance is limited. Therefore, the purpose
 of this study is to investigate the maintenance of weight loss and immunometabolic health
 outcomes after diet-induced weight loss followed by one-year treatment with a glucagon-like
 peptide-1 receptor agonist (liraglutide), physical exercise, or the combination of both
 treatments as compared with placebo in individuals with obesity.

 Methods and analysis: This is an investigator-initiated, randomized, placebo-controlled,
 parallel group trial. The investigators will enroll women and men (age 18 to 65 years) with
 obesity (body mass index 32 to 43 kg/m2) to adhere to a very low-calorie diet (800 kcal/day)
 for eight weeks in order to lose at least 5 % of body weight. Subsequently, participants will
 be randomized in a 1:1:1:1 ratio to one of four study groups for 52 weeks: 1) placebo, 2)
 exercise 150 min/week + placebo, 3) liraglutide 3.0 mg/day, and 4) exercise 150 min/week +
 liraglutide 3.0 mg/day. Re-screening is allowed within the recruitment period.

 The primary endpoint is change in body weight from randomization to end-of-treatment.

 Ethics and dissemination: The trial has been approved by the ethical committee of the Capital
 Region of Denmark (H-16027082) and the Danish Medicines Agency (EudraCT 2015-005585-32). The
 trial will be conducted in agreement with the Declaration of Helsinki and monitored to follow
 the guidelines for good clinical practice. Results will be submitted for publication in
 international peer-reviewed scientific journals.
Obesity is associated with increased risk of developing cardiovascular disease and type 2
 diabetes (T2D), along with increased risk of all-cause mortality [1,2]. Obesity management
 guidelines recommends weight loss of more than 5 % of initial body weight to improve
 cardiometabolic risk factors, with greater weight loss producing greater benefits [3,4]. A 5
 to 10 % weight loss improves lipid profile (~20% reduction in triglycerides, ~15 % reduction
 in LDL-cholesterol, ~8 % increase in HDL-cholesterol levels) [1,4,5], reduces systolic and
 diastolic blood pressure (~5 and ~4 mmHg, respectively) [3,6], reduces HbA1c [3,4], and
 improves insulin sensitivity [7-9]. However, weight regain reverse these health benefits
 [10,11]. Furthermore, intentional weight loss is typically followed by a 30 to 50 % regain of
 lost weight within the first year [12-14]. The main biological reasons for the rapid weight
 regain may be that weight loss causes a decrease in total energy expenditure to a degree that
 is greater than predicted from the decrease in fat and lean mass [15,16] in combination with
 increased appetite in the weight-reduced state [17,18].

 Increasing energy expenditure by increasing physical activity is the first-line lifestyle
 modification in the treatment of obesity along with reducing food intake. For exercise
 interventions targeting general public health recommendations (at least 150 min/week of
 moderate intensity aerobic exercise), the associated weight loss is often modest (0-3 %)
 without concomitant calorie restriction [19-21]. However, independent of weight loss,
 increasing physical activity improves body composition, glycemic control, low grade
 inflammatory profile, and cardiorespiratory fitness in individuals with overweight and
 obesity [22-25]. In addition, exercise may preserve lean mass during weight loss [26] and
 thereby counteract the associated decrease in resting metabolic rate [27], which may explain
 the observation that individuals performing regular exercise have less body weight regain
 after weight loss compared to participants that do not exercise [28,29].

 Glucagon-like peptide-1 (GLP-1) is an incretin hormone primarily secreted from
 enteroendocrine L-cells in the gut after food intake. GLP-1 stimulates glucose-dependent
 insulin secretion thereby lowering blood glucose and reduces appetite and thereby food intake
 [30,31]. Treatment for 56 weeks with the GLP-1 receptor agonist (GLP-1 RA), liraglutide (3.0
 mg), as an adjunct to regular diet and physical activity recommendations has been shown to
 improve glycemic control and induce moderate weight loss of 4.0 % in patients with T2D [32]
 and 5.4 % in non-diabetic individuals with overweight or obesity [33] compared to placebo. In
 addition, liraglutide has been shown to maintain a diet-induced weight loss over 56 weeks
 [34] and maintain very low-calorie diet-induced improvements of fasting plasma glucose and
 triglycerides over 52 weeks of weight loss maintenance superior to similar diet-induced
 weight loss maintenance in obese nondiabetic individuals [18].

 Obesity is associated with chronic low-grade inflammation [35,36] which is linked to the
 development of atherosclerosis and insulin resistance [37-39]. Physically active individuals
 have lower inflammatory biomarker concentrations than their inactive counterparts [24],
 possibly explained by antiinflammatory effects of an acute bout of exercise [40] and lower
 levels of visceral adipose tissue [41]. GLP-1 has also emerged as an immunomodulatory agent
 [42,43]. In mice, GLP-1 RA administration reduces macrophage accumulation and inflammatory
 markers in the arterial wall [44], adipose tissue [45], and heart [46]. Similarly, GLP-1 RAs
 have shown antiinflammatory effects in human coronary artery endothelial cells and aortic
 endothelial cells [47]. In humans with T2D, short term GLP-1 RA treatment exert
 antiinflammatory actions, reflected in reduced levels of the macrophage activation molecule
 sCD163 [48] and reduced production of several proinflammatory markers, such as TNF-α, IL1β,
 and IL-6 in peripheral blood mononuclear cells [48,49]. Another study showed no improvement
 of obesity-associated adipose tissue dysfunction in T2D patients after GLP-1RA treatment
 [50]. One year treatment with GLP-1 RAs reduce the inflammation marker, high-sensitivity
 C-reactive protein, in overweight and obese individuals [33] and T2D patients [51]. Notably,
 in patients with T2D and high cardiovascular risk, GLP-1 RAs reduced the rate of occurrence
 of first major cardiovascular event [52,53].

 Thus, both physical activity and GLP-1 RA treatment seem to facilitate weight loss
 maintenance, improve metabolic health, and reduce systemic inflammation. However,
 diet-induced weight loss decreases energy expenditure and increases appetite. The
 investigators hypothesize that the combination of physical activity and liraglutide treatment
 improves weight loss maintenance and immunometabolic health since the decreased energy
 expenditure is targeted with exercise and the increased appetite with liraglutide.

 Objective:

 The objectives of this study are to investigate the maintenance of weight loss and
 immunometabolic health outcomes over 52 weeks with liraglutide treatment, physical exercise,
 and the combination in individuals with obesity, after a very low-calorie diet.

 Endpoints:

 Primary endpoint: The primary endpoint is change in body weight from after the initial weight
 loss phase (baseline/V1) to end of treatment after 52 weeks (end/V3).

 Secondary endpoints: The secondary endpoints are changes in a) body composition (fat %, lean
 and fat mass ) and b) metabolic health (glucose tolerance (HOMA-IR, Matsuda, HbA1c), lipid
 status, waist circumference, blood pressure) from V1 to V3.

 Other prespecified endpoints:

 Prespecified endpoints include changes from V0 to V1 to V3 in the following parameters:

 -  Physical fitness (measured by VO2 peak test on a bike, strength test, and functional
 stair test)

 -  Fasting and meal-related hormonal response (e.g. GLP-1, PYY, ghrelin, leptin, etc.)

 -  Food preferences and subjective appetite sensation (measured by the Leeds Food
 Preference Questionnaire (LFPQ) and visual analogue scales)

 -  Determination of daily physical activity and sleep (measured by triaxial accelerometry
 (GENEActiv, ActivInsights Ltd, UK) and questionnaires (PSQI and IPAQ)).

 -  Endothelial function (e.g. measured by flow-mediated dilation (FMD) and relevant
 systemic biomarkers)

 -  Standard clinical blood samples (e.g. C-reactive protein, vitamin D, and glycated
 hemoglobin)

 -  Electrocardiogram (ECG)

 -  Heart rate

 -  Health-related questionnaires (self-rated quality of life (The Short Form (36) Health
 Survey), eating habits (three-factor eating questionnaire), physical activity
 (International Physical Activity Questionnaire), sleep quality (Pittsburgh Sleep Quality
 Index), and self-efficacy (General Self-Efficacy Scale).

 -  Systemic markers of immunometabolism (e.g. measured by single cell analysis)

 -  Immunometabolic changes in the subcutaneous adipose tissue (e.g. measured by single cell
 analysis)

 -  Gene expression profile of circulating inflammatory cells (e.g. in adipose tissue cells
 in which proinflammatory and antiinflammatory adipocytokines, adipocyte differentiation
 markers, and markers of macrophages infiltration will be determined by reverse
 transcription-qPCR).

 -  Bone health (measured by DXA scan and relevant systemic bone markers)

 -  Faecal bacterial composition

 -  Metabolomics and proteomics

 -  Epigenetics of spermatozoa

 -  Use of medication

 -  Proportion of participants with 5,10, 15 and 20% total weight loss

 -  Weight loss from V0-V3

 Follow-up visit 1 year after end of treatment:

 -  Body weight, height, waist and hip circumference, blood pressure and pulse

 -  Fasting blood samples (identical to samples obtained for V0)

 -  DEXA scan

 -  Questionnaires as in V0

 -  Accelerometer device (GENEActiv) worn on wrist for 7 consecutive days and nights after
 follow-up testing to assess physical activity levels
Inclusion Criteria:

 -  BMI > 32 and < 43 (kg/m2)

 -  Age >18 and <65 years

 -  Safe contraceptive method

 Exclusion Criteria:

 -  Patients diagnosed with known serious chronic illness including type 1 or 2 diabetes
 (or a randomly measured fasting plasma glucose > 7 mmol/l)

 -  Angina pectoris, coronary heart disease, congestive heart failure (NYHA III-IV)

 -  Severe renal impairment (creatinine clearance (GFR) <30 mL/min)

 -  Severe hepatic impairment

 -  Inflammatory bowel disease

 -  Gastroparesis

 -  Cancer

 -  Chronic obstructive lung disease

 -  Psychiatric disease, a history of major depressive or other severe psychiatric
 disorders

 -  The use of medications that cause clinically significant weight gain or loss

 -  Previous bariatric surgery

 -  A history of idiopathic acute pancreatitis

 -  A family or personal history of multiple endocrine neoplasia type 2 or familial
 medullary thyroid carcinoma

 -  Osteoarthritis which is judged to be too severe to manage the exercise programme. As
 intended per study design the intervention will include a 5% weight loss prior to
 randomization, thus it is expected that possible participants with mild form of
 osteoarthritis will be able to manage exercise prescriptions.

 -  Pregnancy, expecting pregnancy or breast feeding. If a study participant is in doubt
 whether she could be pregnant, a urine pregnancy test is performed. Females of
 childbearing potential who are not using adequate contraceptive methods (as required
 by local law or practice). Adequate contraception must be used throughout the study
 period and at least 65 hours after discontinuation of trial medication (65 hours
 corresponds to 5 times the half-life of Saxenda). Allergy to any of the
 ingredients/excipients.

 -  Allergy to any of the ingredients/excipients of the study medication: liraglutide,
 disodium phosphate dihydrate, propylene glycol, phenol, hydrochloric acid, sodium
 hydroxide.

 -  Regular exercise training at high intensity (e.g. spinning) >2 hours per week.