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.

The above-mentioned are masked in terms of Liraglutide/placebo, not in terms of exercise/non-exercise.

Statistical analysis of primary outcome will be blinded to the assessor.