Dietary Protein and Monocyte/Macrophage Mammalian Target of Rapamycin (mTOR) Signaling

Study Overview
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Brief Summary
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High protein low carbohydrate diets have become popular in recent years to help facilitate weight loss. It is controversial if these diets are associated with an increased risk of cardiovascular disease. The investigators propose to administer high and low protein shakes to participants and measure effects on circulating monocytes, immune cells critical to the development of atherosclerosis and cardiovascular disease. In order to study circulating monocytes, blood will be collected from the study participants just prior to drinking the shake, and then 1 and 4 hours after drinking the shake. In order to assess functional effects on monocytes, investigators will perform a series of assays comparing the results between individuals who drank high protein vs low protein shakes.

Detailed Description
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Cardiovascular disease remains the leading cause of death globally with obesity as of one of the dominant modifiable risk factors. Obesity is also a precursor to several other cardiovascular risk factors including hypertension, hyperlipidemia, and diabetes. Almost all weight loss efforts utilize dietary modification with high protein/low carbohydrate diets serving as one of the most popular approaches. Despite the metabolic benefits of high dietary protein, recent studies have raised a concerning association with increased risk of atherosclerosis and cardiovascular disease. Although this remains controversial, there is some animal data showing evidence of dietary protein's proatherogenic role. These data are correlative and no mechanistic studies have been undertaken. The downstream events after protein ingestion involve digestion of the protein into amino acids, increases in blood amino acids, and distribution to target tissues. Mouse models have definitively shown that circulating monocytes and macrophages of arterial blood vessels are particularly sensitive to this amino acid load with robust activation of the mTOR (mammalian target of rapamycin) signaling pathway. This in turn leads to inhibition of essential degradative processes of the macrophage such as autophagy and promotes release of pro-inflammatory cytokines. Thus, macrophage function in vascular beds becomes pathogenic leading to atherogenesis and cardiovascular disease The translation of these mechanistic studies in animal models to human is the next obvious step in this research. However, no studies have elucidated the mechanisms of monocyte activation and function following administration of high dietary protein in humans. The investigators propose a pilot study to bridge an important gap in translational research which will elucidate the mechanisms by which dietary protein affects human monocyte function and the risk of atherosclerotic plaque formation. Specifically, the investigators will evaluate the acute activation of mTOR signaling and downstream sequelae in circulating monocytes following the administration of protein shakes. This study will address the hypothesis that humans exposed to high dietary protein will have significantly higher post-prandial monocyte mTOR activation with concomitant development of impaired degradative capacity and a proinflammatory state. An understanding of these mechanisms has broad implications in the evaluation and future therapeutic interventions of cardiovascular disease. In addition, this can provide a valuable clinical tool for health care providers in educating patients on dietary changes to ameliorate cardiovascular risk.

Official Title
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Acute Effects of Dietary Protein on Monocyte/Macrophage mTOR Signaling and Downstream Sequela

Conditions
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Dietary Protein

Intervention / Treatment
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* Dietary Supplement: Dietary protein shake


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: 18+ years of age Able to drink milk based protein shake Exclusion Criteria: Current Pregnancy Any food allergies Personal Hx of Diabetes Personal Hx of Heart Disease Personal Hx of High blood pressure Personal Hx of Stroke Personal Hx of Cancer Personal Hx of Organ transplant Taking Rapamycin/Sirolimus Taking Torisel/Temsirolimus Taking Afinitor/Everolimus Taking any statin medication (eg.simvastatin/atorvastatin/rosuvastatin etc)

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Non-Randomized
Intervention Model: Crossover Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: High protein<br>Subjects getting high protein shake | Dietary Supplement: Dietary protein shake<br>* It is a milk based protein shake. Ingredients include a combination of the following depending on protein content: Boost Plus (a commercial supplement), Unjury (a commercial whey protein isolate), nonfat dry milk powder, Sol Carb (commercial supplement composed of a carbohydrate polymer), canola oil, and water. In order to ensure consistency across all participants each beverage will be prepared in the Clinical Translational Research Unit Metabolic Kitchen under the supervision of a registered dietitian prior to the study participant's visit. Ingredients are individually weighed on a food scale by metabolic kitchen staff to the nearest 0.1 g and then mixed using a magnetic stir plate. Nutritional breakdown of the smoothies (high versus low protein): High protein drink nutrition: 500 kcal per serving, 50% protein, 17% fat, and 36% carbohydrate. Low (standard) protein drink nutrition: 500 kcal per serving, 10% protein, 17% fat, and 73% carbohydrate.<br>|
| Active Comparator: Low protein<br>Subjects getting low protein shake | Dietary Supplement: Dietary protein shake<br>* It is a milk based protein shake. Ingredients include a combination of the following depending on protein content: Boost Plus (a commercial supplement), Unjury (a commercial whey protein isolate), nonfat dry milk powder, Sol Carb (commercial supplement composed of a carbohydrate polymer), canola oil, and water. In order to ensure consistency across all participants each beverage will be prepared in the Clinical Translational Research Unit Metabolic Kitchen under the supervision of a registered dietitian prior to the study participant's visit. Ingredients are individually weighed on a food scale by metabolic kitchen staff to the nearest 0.1 g and then mixed using a magnetic stir plate. Nutritional breakdown of the smoothies (high versus low protein): High protein drink nutrition: 500 kcal per serving, 50% protein, 17% fat, and 36% carbohydrate. Low (standard) protein drink nutrition: 500 kcal per serving, 10% protein, 17% fat, and 73% carbohydrate.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Determination of amino acid levels and mTOR activation in circulating monocytes isolated from subjects ingesting high vs low protein drinks. | Changes in amino acid levels and corresponding changes in mTOR activation will be quantified at baseline (time 0 hour prior to ingestion of a protein shake), then at 1 and 3 hours after ingestion of a protein shake. | 0 Hour, 1 Hour, and 3 Hours | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Determination of changes in autophagy and apoptosis markers in circulating monocytes over time after ingestion of a protein shake | Blood collected at three different time points (0, 1 and 3 hours) will be used to measure changes in markers of autophagy and apoptosis at baseline (time 0 hour prior to ingestion of a protein shake), then at 1 and 3 hours after ingestion of a protein shake. | 0 Hour, 1 Hour and 3 Hours | 
| Determination of changes in reactive oxygen species in circulating monocytes over time after ingestion of a protein shake. | Blood collected at three different time points (0, 1 and 3 hours) will be used to measure changes in levels of reactive oxygen species at baseline (time 0 hour prior to ingestion of a protein shake), then at 1 and 3 hours after ingestion of a protein shake. | 0 Hour, 1 Hour, and 3 Hours | 
| Determination of changes in inflammatory markers in circulating monocytes over time after ingestion of a protein shake. | Blood collected at three different time points (0, 1 and 3 hours) will be used to measure changes in cytokines at baseline (time 0 hour prior to ingestion of a protein shake), then at 1 and 3 hours after ingestion of a protein shake. | 0 Hour, 1 Hour, and 3 Hours | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Atherosclerosis, Macrophage/monocyte, mTOR, Autophagy, Apoptosis, Inflammation