Testing protein leverage in lean humans: a randomised controlled experimental study

Abstract

A significant contributor to the rising rates of human obesity is an increase in energy intake. The 'protein leverage hypothesis' proposes that a dominant appetite for protein in conjunction with a decline in the ratio of protein to fat and carbohydrate in the diet drives excess energy intake and could therefore promote the development of obesity. Our aim was to test the 'protein leverage hypothesis' in lean humans by disguising the macronutrient composition of foods offered to subjects under ad libitum feeding conditions. Energy intakes and hunger ratings were measured for 22 lean subjects studied over three 4-day periods of in-house dietary manipulation. Subjects were restricted to fixed menus in random order comprising 28 foods designed to be similar in palatability, availability, variety and sensory quality and providing 10%, 15% or 25% energy as protein. Nutrient and energy intake was calculated as the product of the amount of each food eaten and its composition. Lowering the percent protein of the diet from 15% to 10% resulted in higher (+12±4.5%, p = 0.02) total energy intake, predominantly from savoury-flavoured foods available between meals. This increased energy intake was not sufficient to maintain protein intake constant, indicating that protein leverage is incomplete. Urinary urea on the 10% and 15% protein diets did not differ statistically, nor did they differ from habitual values prior to the study. In contrast, increasing protein from 15% to 25% did not alter energy intake. On the fourth day of the trial, however, there was a greater increase in the hunger score between 1-2 h after the 10% protein breakfast versus the 25% protein breakfast (1.6±0.4 vs 25%: 0.5±0.3, p = 0.005). In our study population a change in the nutritional environment that dilutes dietary protein with carbohydrate and fat promotes overconsumption, enhancing the risk for potential weight gain.

Figure 1. Lean humans increase energy consumption on a lower percent protein diet.

(A) Cumulative daily bi-coordinate means for protein and non-protein macronutrient (carbohydrate and fat) intake (MJ) for participants during the 4-day 10% (white circles), 15% (grey triangles) and 25% (black squares) ad libitum study periods. The dashed lines represent the nutrient rails participants were restricted to during the 10%, 15% and 25% study periods. The dotted lines represent intakes that may occur on the 10%, 15% and 25% foods if intake was regulated to energy requirements (calculated as 1.6× basal energy requirements as derived from the Schofields equation assuming a light to moderate level of physical activity) and that protein and carbohydrate were interchangeable. The inset shows total energy intake (MJ) for participants over the 4-day 10% (white), 15% (grey) and 25% (black) ad libitum study periods. The same letter above the bars in the insert indicates that the means did not differ significantly in Bonferroni post hoc comparisons, whereas different letters indicate differences at p<0.05. Refer to Figure S1 for individual total energy intake data points. (B) Daily protein (triangles) and total (circles) intake (MJ) for participants during the 4-day 10% (white), 15% (grey) and 25% (black) ad libitum study periods. Intake did not change statistically across days within each treatment. Note that the appearance of an increase in intake (of all nutrients, due to fixed diet compositions) from days 1 to 2 and a decline from day 3 to 4 reflected commencement of the study after breakfast on day 1 and fasting overnight on day 4 in readiness for a meal test on day 5 (data not reported). (C) Cumulative protein (triangles) and total (circles) intake (MJ) for participants during the 4-day 10% (white), 15% (grey) and 25% (black) ad libitum study periods.

Figure 2. Sweet and savoury intake during the 4-day ad libitum period.

Total (triangle), anytime (square) and meal-time (circle) savoury (A) and sweet (B) intake (MJ) during the 4-day ad libitum 10% (white), 15% (grey) and 25% (black) protein study periods. Bi-coordinate means for ‘anytime’ and ‘meal time’ savoury (C) and sweet (D) foods as a percent of total intakes for participants over the 4-day 10% (white circles), 15% (grey triangles) and 25% (black squares) ad libitum study periods. Pairwise comparison performed with Bonferroni post hoc comparisons - **10% significantly different to 15% and 25%, p<0.05; * 10% significantly different to 25%, p<0.05.

Figure 3. Hunger and fullness scores on 10%, 15% and 25% protein diets.

On study day 4, subjects completed a visual analogue scale (VAS) questionnaire to assess subjective hunger and fullness. The VAS questionnaire asked subjects to assess their hunger by reference to a 10 cm horizontal line anchored at one end with the extreme feeling “not at all hungry” and at the other end with “very hungry” and fullness with the extreme feeling “not at all full” and at the other end “very full”. This was done hourly from before breakfast until 10pm. Participants were free to consume breakfast anytime between 08:00–10:00, after which lunch and dinner times were fixed. Hunger and fullness curves have been plotted accordingly. The score prior to breakfast (first of the day) and the 2 scores following breakfast were plotted for hunger (A) and fullness (D). From 12:00 onwards scores for hunger (B) and fullness (D) were plotted on the hour. Figures 3 C and F show the increase in hunger and decrease in fullness from 1 to 2 h following breakfast. Bars with different letters are significantly different (P<0.05) with Bonferroni post hoc comparisons.

Figure 4. Protein leverage study foods.

The 3 photos on the left column are the 10%, 15% and 25% versions (top to bottom) of each food given to participants at breakfast on study day 2. In the right hand column the three photos are the 10%, 15% and 25% versions (top to bottom) of each food given to participants at dinner on study day 2. Participants were offered a set amount of each food that was the same on each study period. The plates were the same for a particular food on each study period.

Figure 5. Palatability of foods served on each 4-day ad libitum period.

Pleasantness (A), savouriness (B) and sweetness (C) ratings given to the 10% (white), 15% (grey) and 25% (black) versions for the 12 foods offered on study day 4: 1. Apricot, walnut and yoghurt muesli, 2. Pear, Raspberry and Coconut Bread, 3. Savoury Breakfast Muffins, 4. Pasta Salad, 5. Sweet Potato and Ricotta Wrap, 6. Apple Crumble Muffins, 7. Hokkien Noodles, 8. Massaman Curry, 9. Chocolate, Apple and Ricotta Cake, 10. Custard, using visual analogue scales scored from 0 to 10 cm.