Metabolism of Fibrinogen and Apolipoprotein B-100 in Childhood Obesity and Cardiovascular Disease

Since obesity and plasma fibrinogen levels are important CVD risk factors in the adults, and since childhood obesity is a major risk factor for adult obesity and also because it is not established whether or not this is due to an increase in the FSR of fibrinogen, the investigators set up the studies with the following specific aims: To investigate the metabolism of fibrinogen and VLDL apoB-100, CVD risk factors, in childhood obesity by measuring their fractional synthetic rate (FSR) compared to lean age and sex matched controls To determine the outcome of a three month non-pharmacological intervention (physical exercise combined with controlled diet) to reduce weight on the FSR of fibrinogen and apoB-100 To determine the relationship between FSR of fibrinogen and IL-6 in obese children and its potential implications on CVD before and after the non-pharmacological intervention To determine other CVD risk factors, PAI-1 levels, D-Dimer concentration, homocysteine, insulin, free fatty acid, HDL & LDL cholesterol and blood pressure in response to weight reduction (as consequence of a combined program of diet and exercise).

A. SPECIFIC AIMS: Evidence from the literature suggests that: childhood onset of obesity increases the risk of obesity and cardiovascular disease (CVD) in adulthood; plasma fibrinogen and apolipoprotein B-100 (apoB-100) levels are elevated in obesity; elevated levels of plasma fibrinogen and apolipoprotein B-100 (apoB-100) are major independent risk factors for CVD in adults; raised plasma fibrinogen levels in obese children and/adolescents could be a major factor responsible for CVD morbidity and mortality in adulthood; a low level of physical activity is associated with a high level of plasma fibrinogen in adults; physical training and controlled diet have been proved to be non-pharmacological ways to improve hemostatic function in adults, thereby reducing heart disease risk. Despite these evidences the mechanism of these changes remain unclear. A better understanding of the mechanism of these changes will lead to more directed therapies to reduce these risk factors early in life. The increased plasma concentrations of fibrinogen (and/or other proteins) is decided by the equilibrium between two dynamic processes in the body, namely, synthesis and degradation rates of these proteins. Therefore, elevated levels of fibrinogen must be a consequence of: increased fibrinogen synthesis; decreased fibrinogen degradation or a contribution of both. For designing effective therapies it is important to know, whether the increased concentration of plasma fibrinogen is due to increased synthesis or decreased degradation or both. Using stable isotope mass spectrometry techniques it is possible to measure the synthesis rates of proteins in vivo in humans. Yet until now there are no reports on the direct measurement of FSR of fibrinogen and/or apoB-100 in childhood obesity, important CVD risk factors. The direct measurement of degradation rates of proteins in vivo in humans, however, is not easily done. Few studies have reported changes in the concentrations of fibrinolytic markers such as plasminogen activator inhibitor-1 (PAI-1) and D-Dimer in obesity. This, however, accounts for only a small fraction of the elevated levels of plasma fibrinogen in childhood obesity. On the other hand recent preliminary results in three subjects from our lab show that fibrinogen synthesis rate is substantially higher (more than two times) in obese adolescent girls Vs lean controls (please see results under "Preliminary results" below). Pro-inflammatory cytokines, particularly interleukin-6 (IL-6), are known to play an important role in the regulation of acute phase reactive proteins associated with inflammation, including fibrinogen. A direct link between IL-6 and increased FSR of plasma fibrinogen in childhood obesity and CVD, however, is unknown. Also, other mechanisms involving insulin resistance and free fatty acid/albumin ratio are also equally plausible for the increased synthesis of fibrinogen in obesity and CVD. Therefore, it is important to understand and establish the relationship between increased FSR of fibrinogen and these regulating factors in this study for two reasons: to better design effective therapies based on these results and to further understand the pathophysiology of these changes in CVD and obesity. The overall purpose of the proposed studies is therefore: to expand on our preliminary data and establish the observation of increased fibrinogen synthesis rate (in three subjects) in childhood obesity to understand the relationship between increased FSR of fibrinogen and changes IL-6, insulin and glucose levels and free fatty acid (FFA) levels and FFA/albumin ratio in obese vs lean children to study the effect of a non-pharmacological intervention program for three months (12 weeks), that involves weight reduction due to exercise and controlled diet on fibrinogen, apoB-100 and other cardiovascular risk factors and their relationship to changes in IL-6 levels, insulin/glucose ratio (a measure of insulin resistance), FFA/albumin ratio. The proposed grant will use in vivo stable isotope (non-radioactive) dilution techniques, to test the following hypothesis: Hypotheses: higher rates of synthesis of fibrinogen and apoB-100 contribute substantially to their elevated levels in obese children; plasma fibrinogen and apoB-100 concentration and synthesis decrease by non-pharmacological intervention to reduce weight, that involves regular physical exercise and controlled diet; IL-6 and/or insulin resistance are important mechanistic links between increased plasma fibrinogen FSR and increased CVD risk and obesity and the non-pharmacological intervention program modulates other CVD risk factors such as PAI-1, D-Dimer, homocysteine, free fatty acids, insulin, glucose, HDL and LDL cholesterol levels and IL-6 concentrations along with fibrinogen synthesis. Summary of Specific Aims Since obesity and plasma fibrinogen levels are important CVD risk factors in the adults, and since childhood obesity is a major risk factor for adult obesity and also because it is not established whether or not this is due to an increase in the FSR of fibrinogen, the investigators set up the studies with the following specific aims: To investigate the metabolism of fibrinogen and VLDL apoB-100, CVD risk factors, in childhood obesity by measuring their fractional synthetic rate (FSR) compared to lean age and sex matched controls To determine the outcome of a three month non-pharmacological intervention (physical exercise combined with controlled diet) to reduce weight on the FSR of fibrinogen and apoB-100 To determine the relationship between FSR of fibrinogen and IL-6 in obese children and its potential implications on CVD before and after the non-pharmacological intervention To determine other CVD risk factors, PAI-1 levels, D-Dimer concentration, homocysteine, insulin, free fatty acid, HDL & LDL cholesterol and blood pressure in response to weight reduction (as consequence of a combined program of diet and exercise).

Obese subjects will undergo a randomized physical activity-based weight reduction program (12 weeks). The lifestyle intervention will be determined based on an initial evaluation of each subjects' energy expenditure (by indirect calorimetry). Caloric intake will be set to elicit the loss of 1 to 1.5 lbs/week. The subjects and the family will meet with a dietitian once/week for 12 weeks. The intervention group will undergo an aerobic exercise program 3 times/week. The duration of exercise will be progressively increased from 20 to 45 minutes/session. The subjects will keep daily record of exercise activity and food intake. Interventions will be monitored during the subjects' weekly visit with the research group and they will be given counseling to stick to the intervention program.

Inclusion Criteria: Obese: BMI ≥30 kg/m2 Lean (controls): BMI ≤ 25 kg/m2) age 14 to 18 years and Tanner stage matched Ability to understand and cooperate with the procedures Signed informed consent from subjects and parents Exclusion Criteria: Medications such as Beta-adrenergic blockers, steroids and other drugs known to affect protein metabolism Heart disease Chronic liver disease Chronic renal disease Active malignancy Alcoholism or drug abuse Anemia Inter-current illness over the 7 days before the study Surgery in the past 3 months

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Cosentino RG, Churilla JR, Josephson S, Molle-Rios Z, Hossain MJ, Prado WL, Balagopal PB. Branched-chain Amino Acids and Relationship With Inflammation in Youth With Obesity: A Randomized Controlled Intervention Study. J Clin Endocrinol Metab. 2021 Oct 21;106(11):3129-3139. doi: 10.1210/clinem/dgab538.