This prospective cohort study investigates the effects of tirzepatide versus liraglutide on bone turnover markers and body composition in adults with class 3 obesity, characterised by Body Mass Index (BMI) ≥40 kg/m². Participants will be followed for 6 months with assessments at baseline, 3 and 6. The primary outcome is the change in bone resorption marker C-terminal telopeptide of type I collagen (CTX) at 3 months. Secondary outcomes include changes in body weight, BMI, bone mineral density (BMD), and body composition. The study aims to clarify the differential impact of weight loss achieved through tirzepatide versus liraglutide on bone metabolism and body composition in adults with obesity.

Title of the study Comparative investigation of changes in body composition and bone turnover markers in people with obesity after treatment with tirzepatide versus liraglutide. A prospective cohort study.

Research Hypothesis:

The investigators hypothesize that treatment with tirzepatide versus liraglutide will yield distinct effects on markers of bone turnover and body composition, independent of the magnitude of weight loss due to different molecular mechanisms of action.

Keywords Obesity, Bone Metabolism, Body Composition, Lean mass, Free Fat Mass, Fat Mass, Tirzepatide, Liraglutide, CTX, P1NP, TRAP5b

Introduction Obesity is a growing global health concern, associated with various metabolic disorders, including hypertension, diabetes, metabolic syndrome, non-alcoholic fatty liver disease, sleep apnea and ultimately cardiovascular events. Obesity has also been linked to osteoporosis and high risk of fragility fractures. However, this relationship remains complex, as it involves multiple factors, including mechanical load from body weight, the type and distribution of adipose tissue, imbalances in nutrient intake, and a wide variety of cytokines (adipokines) and hormones secreted by the adipose tissue. On the other hand, rapid and substantial weight loss is associated with a significant reduction in bone mineral density (BMD), osteomalacia and increased risk of fragility fractures. Assessment of bone turnover status can be reliably assessed by circulating levels of bone turnover markers (BTMs) such as the bone resorption markers C-terminal telopeptide of type I (CTX), and tartrate-resistant acid phosphatase (TRAP5b), which evaluates bone resorptive capacity and is not influenced by renal clearance and bone formation markers such as procollagen type 1 N-terminal propeptide (P1NP). Clinical trials have demonstrated that diet-induced weight loss is associated with deterioration of bone metabolism, as indicated by alterations in BTMs and Dual-energy X-ray Absorptiometry (DXA) measurements of BMD. In addition, bariatric management of obesity, particularly malabsorptive surgical modalities, are known to increase the risk of fractures at two years postoperatively. Weight-loss treatments such as liraglutide, the first injectable Glucagon-like peptide-1 (GLP-1) receptor agonist approved for obesity, and tirzepatide, a dual GLP-1/Glucose-dependent insulinotropic polypeptide (GIP) receptor agonist recently approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency for treating obesity, has shown efficacy in weight management but their effects on bone metabolism are not fully understood. Liraglutide administration in diabetic mice improves bone health, by altering the levels of BTMs CTX, osteoprotegerin (OPG), osteocalcin (OC), alkaline phosphatase (ALP), and procollagen type 1 N-terminal propeptide (P1NP), reducing the number of osteoclasts, and improving bone architecture. Similarly, in mice models with glucocorticoid-induced osteoporosis (GIOP) under liraglutide treatment, BMD and bone microarchitecture is improved, with reductions in BTMs such as Tartrate-resistant acid phosphatase type 5b (TRACP-5b) and CTX, and increases in ALP and OPG. A randomized, double-blinded, placebo-controlled clinical trial of 56 individuals with diabetes (mean age: 63 years) showed that liraglutide at a dose of 1.8 mg did not affect bone metabolism despite a reduction in body weight. Another randomized controlled study of 37 women with obesity (mean age 46 years, BMI 34kg/m2) who lost 12% of body weight after a 12-week low-calorie diet and were subsequently randomized to placebo or liraglutide at a dose of 1.2 mg, found that liraglutide increased P1NP levels by 16%, highlighting its positive effects on bone metabolism following weight loss. Nevertheless, clinical data on the effects of liraglutide on bone metabolism are limited. Regarding tirzepatide, the pronounced weight loss from its administration seems to have a neutral effect on bone mass and microarchitecture in diabetic mice, with no significant changes in BTMs. However, no clinical data are currently available regarding tirzepatide effect on bone metabolism. The concern, however, is that the combination of GLP-1 and GIP, which leads to greater weight loss, may have a different impact on bone metabolism than GLP-1 agonists alone.

Necessity/ Originality The available human studies in populations with diabetes and obesity are limited, with most reaching a maximum dose of Liraglutide in 1.8 mg daily and not resulting in significant weight loss associations with bone metabolism. There are no human studies with tirzepatide concerning its impact on bone metabolism.

The necessity and originality of the study lies in the fact that no previous research has investigated the effects of tirzepatide (at maximum doses of 10 -15 mg weekly) and liraglutide (at dose of 3 mg daily) on both body composition parameters and bone metabolism in individuals with obesity.

The aim of the present study is to investigate the potential differential effects of tirzepatide versus liraglutide on bone turnover status and body composition parameters in patients living with obesity and explore the association with differences in the magnitude of weight loss and the different mechanisms of action (GlP-1 agonism versus GIP/GLP-1 synergy).

Primary - Objectives:

1. To evaluate the impact of tirzepatide on bone turnover status and bone mineral density in patients with obesity.
2. To assess the effects of liraglutide on bone turnover status and bone mineral density in patients with obesity.
3. To compare the alterations in bone metabolism across the 2 intervention groups, after adjustments for the differences in weight loss.

Secondary - Objectives:

1. To evaluate the impact of tirzepatide on body composition parameters in patients with obesity.
2. To assess the effects of liraglutide on body composition parameters in patients with obesity.
3. To compare the alterations in body composition parameters across the 2 intervention groups, after adjustments for the differences in weight loss.

Significance of the Study/ Clinical relevance This research will provide valuable data regarding the effects of weight-loss medications on bone health and body composition in individuals living with obesity. The findings may influence clinical practice guidelines and therapeutic approaches in managing obesity and its associated complications.

Methodology

1. Study Design:

   Open-label observational cohort study

2. Inclusion Criteria-Participants:

   Adults aged over 30 years with BMI ≥40 kg/m²

3. Exclusion criteria:

   • Type 2 Diabetes Mellitus (T2DM) and type 1 Diabetes Mellitus (T1DM)
   • Chronic kidney disease
   • Liver failure
   • Heart failure
   • Malignancy coexistence
   • Previous bariatric or gastrointestinal surgery involving intestinal bypass
   • Uncontrolled hypo/hyperthyroidism
   • Uncontrolled hypo/hyperparathyroidism
   • Pregnancy and lactation
   • Recent fracture (within 2 years)
   • Rare Metabolic Bone Diseases (e.g., Paget's disease of bone, fibrous dysplasia, osteopetrosis)
   • Inflammatory arthritis
   • Medications which can affect bone markers: bone-anabolic agents, antiresorptive agents, antiandrogenic agents, vitamin K antagonists, antipsychotic agents, contraceptives, glucocorticoids (oral), methotrexate, thiazides, aromatase inhibitors etc)
   • Hemolytic anemia

   Patients who will be deemed eligible for the study and sign the informed consent form will undergo the following research protocol.

4. The step-by-step process of the study Visit 1 Medical history documentation and measurement of anthropometric parameters (Height, Weight, BMI, Waist-to-Hip Ratio, waist to height ratio).

   Assessment of SARC-F questionnaire , Short-Form 36 (SF-36) Health Survey and Perceived Stress Scale - 14 item (PSS-14) (validated for the Greek population).

   Bioelectrical impedance analysis (BIA) Resting Metabolic Rate (RMR). Grip strength, Chair stand test and Gait speed. DXA at three skeletal sites (lumbar spine and both hips), calculation of Trabecular Bone Score (TBS) and Whole-Body Scan.

   Blood sampling*. Hypocaloric diet designed by the same dietary team, providing an energy intake 20-30% below the Total Energy Expenditure, as estimated from the Resting Metabolic Rate and exercise recommendations.

   Patients then will be prescribed liraglutide or tirzepatide based on their discretion and their treating physician's choice.

   Weekly estimation of each participant from Group A - liraglutide Group to assess the potential for a dosage increase starting from an initial dose of 0.6 mg daily, with incremental increases of 0.6 mg, up to the maximum tolerated dose, with a maximum target dose of 3 mg per day.

   Monthly estimation of each patient from Group B - tirzepatide Group to assess the potential for a dosage increase, starting from an initial dose of 2.5 mg weekly, up to the maximum tolerated dose, with a maximum target dose of 10-15 mg per week.

   Visit 2-Follow up in 3rd month Medical history documentation and measurement of anthropometric parameters (Height, Weight, BMI, Waist-to-Hip Ratio, waist to height ratio).

   BIA. Grip strength, Chair stand test and Gait speed. Blood sampling*. Patients continue to receive liraglutide or tirzepatide. Monitoring the adherence to the diet and exercise program based on the patient's reports.

   Visit 3-Follow up in 6th month Medical history documentation and measurement of anthropometric parameters (Height, Weight, BMI, Waist-to-Hip Ratio, waist to height ratio).

   Assessment of SARC-F questionnaire, SF-36 Health Survey and PSS-14 (35) (validated for the Greek population).

   BIA. RMR. Grip strength, Chair stand test and Gait speed. DXA at three skeletal sites (lumbar spine and both hips), calculation of TBS and Whole-Body Scan.

   Blood sampling*. Patients continue to receive liraglutide or tirzepatide. Monitoring the adherence to the diet and exercise program based on the patient's reports.

   * Blood sampling for the determination of CTX, P1NP, TRAPC5b, OC, calcium, phosphate, albumin, ALP, parathyroid hormone (PTH), 25-hydroxyvitamin D [25(OH)D], insulin-like growth factor 1 (IGF1), adiponectin, leptin, and insulin will be conducted during the morning hours between 8 and 10 a.m., following an 8-hour fasting period and avoidance of strenuous exercise the previous day. For premenopausal women, blood sampling will be conducted during the follicular phase of the menstrual cycle, based on the patient's menstrual history.

5. Study Endpoints:

   • The primary endpoint is to investigate comparative changes in the bone resorption marker CTX at 3 months with liraglutide vs tirzepatide.

   • Secondary endpoints include:

     1. changes in body weight and BMI at 6 months
     2. BMD and body composition parameters measured by DXA scan at baseline and after 6 months of intervention.

6. Data Analysis:

   • Statistical analysis will be performed using ANOVA for comparison between groups. Correlation with changes in weight and bone metabolism indices will also be assessed using regression analysis.

7. Sample size calculation The sample size calculation for this study was based on the least significant change (LSC) of the C-terminal telopeptide of type I collagen (CTX) marker in serum at three months. Using this threshold, the investigators determined that a total of 32 patients per arm would be required to achieve adequate statistical power (80% statistical power (false negative), 5% error (false positive). With anticipated drop-out rate of 10%, about 36 participants/ per group should be randomized, ensuring sufficient power to detect meaningful changes in CTX levels while accounting for potential variability in the biomarker response.

8. Recruitment centers Laikο General Hospital, Athens Athens Medical Center Sotiria General Hospital 251 Hellenic Air Force & VA General Hospital Evangelismos Hospital

9. Protocol for Sample Collection and Processing Fasting Status: Blood samples are collected after an overnight fast. Vigorous exercise is avoided on the day prior to sample collection.

   Timing of Collection: Samples are collected between 7:30 a.m. and 10:00 a.m. Sample Type: Serum and EDTA plasma are used. Sample Processing: Samples are centrifuged immediately and serum and plasma separated and stored into aliquots. Hemolysis is avoided during handling.

   Storage Conditions: Samples are stored at ≤-20 °C until analysis. For long-term storage exceeding three months for CTX-I and six months for PINP, samples are maintained at ≤-70 °C.

   Thawing and Preparation: Thawed samples are mixed thoroughly by inversion before analysis. Centrifugation is performed to remove particulates when necessary.

   Consistency in Monitoring: The same sample type and identical handling conditions are maintained for patient monitoring.

   Batch Analysis of Serial Samples: Serial samples are frozen and analyzed collectively within the same batch.

10. Ethics The study will be reviewed and approved by the Ethics Committee of LAIKO General Hospital of Athens and the Ethics Committee of the Medical School of National and Kapodistrian University of Athens, before initiation. It will be conducted in compliance with the ethical principles outlined in the Declaration of Helsinki, ensuring the protection of participants' rights, safety, and well-being. All participants will provide informed consent before enrollment, and the study will adhere to all relevant regulatory and institutional guidelines for clinical research.

11. Processing capabilities:

1. Timeline: Recruitment: 6 months; may be extended up to 12 months Data Analysis: 3 months Authorship: at least 2 years

2. Budget: A detailed budget will be prepared, covering personnel costs, laboratory tests, participant incentives, and data analysis.

Conclusion:

This research aims to fill the gap in knowledge regarding how different interventions for obesity affect bone metabolism, which is crucial for developing comprehensive treatment protocols for people living with obesity.

Inclusion Criteria:

• Adults aged between 30 and 65 years
• BMI ≥40 kg/m²

Exclusion Criteria:

• Type 2 Diabetes Mellitus (T2DM) and type 1 Diabetes Mellitus (T1DM)
• Chronic kidney disease
• Liver failure
• Heart failure
• Malignancy coexistence
• Previous bariatric or gastrointestinal surgery involving intestinal bypass
• Uncontrolled hypo/hyperthyroidism
• Uncontrolled hypo/hyperparathyroidism
• Pregnancy and lactation
• Recent fracture (within 2 years)
• Rare Metabolic Bone Diseases (e.g., Paget's disease of bone, fibrous dysplasia, osteopetrosis)
• Inflammatory arthritis
• Medications which can affect bone markers: bone-anabolic agents, antiresorptive agents, antiandrogenic agents, vitamin K antagonists, antipsychotic agents, contraceptives, glucocorticoids (oral), methotrexate, thiazides, aromatase inhibitors etc)
• Hemolytic anemia

This study involves patients with a body mass index (BMI) greater than 40 who either present for weight loss management at endocrinology or obesity clinics, or who are referred to sleep disorder centers due to obstructive sleep apnea syndrome requiring CPAP therapy, where weight loss intervention is consequently recommended.

Due to participant privacy concerns and institutional policies, individual participant data will not be shared outside the research team