How Animal and Plant Protein Shakes Change the Body's Nutrient and Health Responses

Randomized Crossover Trial of Protein Source and Leucine Supplementation on Metabolic and Immune Signaling Pathways

The goal of this clinical trial is to learn how different protein types affect the body's metabolism and immune system in healthy adults. The main questions it aims to answer are:

Does drinking smoothies with animal-based or plant-based protein change the level of amino acids like leucine in the blood?

Does adding leucine to a plant-based smoothie make the body respond like it does to an animal-based smoothie?

Participants will:

Come to the research clinic for 3 study visits

Drink a high-protein smoothie at each visit (one with animal protein, one with plant protein, and one with plant protein + leucine)

Give blood samples before and 1 and 3 hours after each smoothie

Researchers will look at how the body's metabolism and immune system respond. They are especially interested in a pathway called mTOR, which helps control how cells use nutrients and may be involved in heart disease like atherosclerosis.

All smoothies are prepared to meet Halal and Kosher dietary guidelines.

This is a randomized, controlled, crossover clinical trial designed to investigate the acute effects of dietary protein source and leucine supplementation on circulating amino acids and downstream metabolic and immune signaling pathways in healthy adults. The study is being conducted by researchers at the University of Pittsburgh and is based on emerging evidence that dietary protein composition-particularly the type and amount of amino acids such as leucine-may influence key biological processes associated with cardiometabolic health.

Proteins are an essential part of the human diet, and different protein sources (plant vs. animal) contain varying amounts and types of amino acids. One amino acid of particular interest is leucine, a branched-chain amino acid (BCAA) known to activate the mTOR (mechanistic target of rapamycin) signaling pathway. The mTOR pathway plays a critical role in regulating cell growth, metabolism, immune responses, and energy use. Excessive or chronic activation of this pathway has been implicated in cardiometabolic diseases, including atherosclerosis, obesity, insulin resistance, and inflammation.

Animal-derived proteins such as milk or meat tend to be richer in leucine than plant-derived proteins like soy or pea protein. This difference may explain some of the metabolic disparities observed between diets high in animal protein versus plant protein. While plant-based diets are often recommended for their anti-inflammatory and cardiovascular benefits, they may lack sufficient leucine to activate anabolic signaling pathways in certain tissues. It remains unclear whether supplementing plant-based protein with free leucine can achieve the same biological responses as animal protein ingestion.

The trial includes three study arms: (1) a smoothie containing only plant-based protein, (2) a smoothie containing animal-based protein, and (3) a plant-based smoothie with added free leucine. Each smoothie is isocaloric and matched in macronutrient content, differing only in protein source and leucine content. All meals will be prepared and administered under controlled conditions at the Clinical and Translational Research Center (CTRC) at UPMC Montefiore.

Participants will be healthy adults without chronic illnesses or recent infections, aged 18 years and older. Each participant will complete all three study visits in a randomized order, with a minimum 3-day washout period between visits. At each visit, participants will be asked to fast for 10 hours overnight before arriving at the CTRC in the morning. Upon arrival, fasting blood will be collected, followed by consumption of the assigned test smoothie. Additional blood samples will be collected at 1 and 3 hours post-consumption to evaluate postprandial responses.

Blood samples will be analyzed for:

Circulating amino acid levels (primary: leucine)

mTORC1 signaling activity in peripheral blood mononuclear cells (PBMCs), assessed via phosphorylation of S6 kinase (pS6)

Inflammatory and immune signaling markers (e.g., IL-6, NF-κB)

Exploratory transcriptomic profiling and broader amino acid analysis if resources allow

The main hypotheses are:

Animal-based protein will induce higher postprandial leucine levels and stronger mTORC1 activation than plant-based protein.

Supplementation of free leucine in the plant-based smoothie will mimic the biological effects of the animal protein smoothie on immune/metabolic responses.

The findings from this study could provide novel insights into how different dietary proteins modulate nutrient sensing and immune pathways. If plant-based protein supplemented with leucine proves to mimic the effects of animal protein, this could support more sustainable and heart-healthy dietary recommendations without compromising key metabolic functions.

Ultimately, this study will contribute to a deeper understanding of the nutritional regulation of mTOR and inflammation in humans and may inform future dietary interventions aimed at preventing or mitigating chronic cardiometabolic diseases such as atherosclerosis and diabetes.

Inclusion Criteria:

1. Age 18-65 years
2. BMI 18.5-29.9 kg/m²
3. Generally healthy, without known chronic illness and/or treatment with chronic medications
4. Willing to comply with dietary restrictions and study procedures
5. Not currently following a prescribed or restrictive diet (for example, vegan, ketogenic or high-protein regimen) or regularly taking high-protein supplements

Exclusion Criteria:

1. History of cardiovascular disease, diabetes, or autoimmune disorders
2. Active use of anti-inflammatory, immunomodulatory, or lipid-lowering medications
3. Renal or hepatic dysfunction
4. Pregnancy or breastfeeding
5. Allergies or intolerances to components of study meals. A list of the smoothie ingredients will be sent to the potential participant with the mailing of the consent form
6. Participation in another interventional study within the last 30 days