DIETARY PROTEIN INTAKE AND MUSCLE GENE EXPRESSION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0196626
• Grant No.: 2003-35200-13779
• Proposal No.: 2003-00858
• Project Start Date: Sep 1, 2003
• Project End Date: Aug 31, 2006
• Grant Year: 2003
• Program Code: [31.0]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
FOOD & NUTRITION

Non Technical Summary
The dietary protein needs of older persons are not known with confidence and inaduate protein intake causes the loss of skeletal muscle mass. This project will use the muscle biopsy and DNA microarray techniques to establish in older women if there is a molecular fingerprint in muscle that identifies whether a person is consuming an adequate or inadequate protein diet.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	5010	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
5010 - Food;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

human nutrition

skeletal muscle

diet

nitrogen balance

elderly

women

dna

adaptation

dietary proteins

single sequence repeats

nutrient intake

recommendations

nutrient requirements

dietary levels

protein intake

gene expression

food

body composition

muscle properties

dna fingerprinting

Goals / Objectives
The current Recommended Dietary Allowance (RDA) for protein is 0.8 grams of protein per kilogram body weight per day for healthy adults of all ages. Older people (age greater than 50 years) often experience metabolic and physiological changes that may alter dietary protein nees, including progressive changes in body composition (especially the loss of skeletal muscle mass), physical activity, and the frequency of disease. Limited nitrogen balance-based research suggests that the protein need of older adults is higher than the current RDA. Other research showed that longer-term adherence to a low-protein diet (56% of the RDA for nine weeks) by older women resulted in negative nitrogen balance and adversely affected lean body mass, muscle mass, and muscle strength and function. Recently published data indicates that long-term adherence to the RDA for protein results in the loss of muscle mass of older men and women. Collectively, these studies document the importance of defining an adequate protein intake for older people to prevent diet-related loss of muscle mass. Since inadequate protein intake adversely affects skeletal muscle, we reason that a dietary protein requirement could be set accurately if one could define the molecular or metabolic profile in muscle that is association with inadequate protein intake.

Project Methods
We propose to use a new and sophisticated research tool called the DNA microarray to study the molecular changes that occur in muscle in response to changes in protein intake. The DNA microarray permits the assessment of the expression of thousands of genes simultaneously. We hypothesize that the profile of gene expression in skeletal muslce will be different in older women when they consume diets with inadequate versus adequate protein. Experimentally, eight women, age range 60-75 years, will consume strictly controlled diets that provide 0.5, 0.75, and 1.0 grams of protein per kilogram body weight per day in three separate four-week trials (cross-over design). Proein requirement will be esteimated using the nitrogen balance techniques. Muscle biopsies will be taken at the end of each trial, the total RNA isolated, and the DNA microarray technique used to profile the gene expression of over 22,500 genes. The results of this research will help establish if there is a molecular fingerprint in muscle that identifies whether a person is consuming and adequate or inadequate protein diet, and will be used as the foundation for future studies of dietary protein adequacy in older people.

Progress 09/01/03 to 08/31/06

Outputs
This project has yielded new and important information regarding the impact of inadequate dietary protein on potentially adverse changes in muscle mRNA levels of young and older adults. Results from an initial study in 21 men and women, age range 55 to 80 years, who consumed strictly controlled diets that contained either 150 percent of the Recommended Dietary Allowance (RDA) for protein for two weeks (adequate protein), or 150 percent of the RDA for one week followed by 63 percent of the RDA for one week (inadequate protein) support that consumption of inadequate protein for one week induces changes in skeletal muscle transcript levels that might precede adverse metabolic, functional, and structural events, including wasting. These changes included differentially expressed transcripts in several functional classess: immune, inflammatory, and stress responses (prediminantly up-regulated); energy metabolism (down-regulated), protein synthesis (down-regulated), and proliferation (down-regulated). A second randomized, cross-over design research study in 12 younger men and 10 older men extend these findings to document numerous age-related changes in skeletal muscle transcript levels. The subjects consumed strictly controlled diets that contained 63 percent, 94 percent, or 125 percent of the RDA for 18-day periods and samples of muscle were obtained on day 12 of each diet period. Analyses using the Microarray Suite 5.0 output has established that three percent of all 54,675 probesets were differentially expressed with dietary protein intake and 6 percent of the total probesets were differentially expressed with age. These findings indicate that dietary protein and age both impact skeletal muscle transcript levels and may contribute to sarcopenia.

Impacts
The results of this research demonstrate that older and younger persons who consume inadequate dietary protein for one to two weeks experience a wide variety of transcript-level changes in skeletal muscle. These responses are consistent with the existence of metabolic and physiological changes that may precede accommodation including inflammatory stress, reduced metabolism and oxygen transport, reduced protein synthesis, and muscle wasting.

Publications

• Thalacker-Mercer AE, JC Fleet, BA Craig, NS Carnell, WW Campbell. Inadequate protein intake affects skeletal muscle transcript profiles in older humans. Am J Clin Nutr (in press, 2007).

Progress 10/01/04 to 09/30/05

Outputs
This project has been approved for a one-year no-cost extension to complete the research. Progress during the past year has been good. While the original aim of the study was to evaluate the effect of dietary protein intake on skeletal muscle gene expression in older women, we have taken advantage of an opportunity to expand the project to also study the effect of age. This was made possible by obtaining muscle samples from groups of 10 older men and 12 younger men who completed three 18-day controlled diet trials. During each trial all of the subjects consumed a protein intake of 0.5, 0.75, of 1.0 grams protein per kg body weight per day. These protein intakes span the range of adequacy, based on the estimated average requirement and recommended dietary allowance of 0.66 and 0.80 g protein per kg body weight per day. Thus, we now have a much larger data set in young and older men that will allow us to accomplish more goals. The data set is now being analyzed to determine the main effects of protein intake and age, and the diet by age interactions, on muscle gene expression in more than twice as many subjects as originally proposed.

Impacts
The results from this study will help evaluate the impact of inadequate protein and chronologial age on skeletal muscle gene expression. We anticipate that diet and age-related differences will be found in several functional groupings of genes related to protein synthesis and degradation, mitochondrial metabolism and energy production, immune function, protein structure, and signaling.

Publications

• No publications reported this period

Progress 10/01/03 to 09/29/04

Outputs
The research is on-going with excellent progress made, but no results to report, yet. Muscle samples obtained from 10 elderly and 12 young subjects who consumed protein intakes of 0.5, 0.75, and 1.0 grams protein per kilogram body weight per day during three 12-day trials (random ordering of protein intakes). The muscle samples were processed to isolate total RNA and the DNA microarray technique was used to profile the gene expression of over 54,000 genes. The gene array data are currently being processed and analyzed.

Impacts
This study will provide a foundation for future prospective studies to assess the effect of inadequate protein intake on skeletal muscle in older people. Inadequate protein intake affects about 40 percent of people above the age of 70 years.

Publications

• No publications reported this period

Progress 10/01/02 to 09/30/03

Outputs
The project was initiated on September 1, 2003, the research team assembled and inital plans for accomplishing the research established. Work is on-going to perfect the RDA isolation methods from skeletal muscle samples. There are no results to report.

Impacts
This study will provide a foundation for future prospective studies to assess the effect of inadequate protein intake on skeletal muscle in older people. Inadequate protein intake affects about 40 percent of people above the age of 70 years.

Publications

• No publications reported this period