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ote that Leucine intake (especially during cardio) in a fasted state is extremely important.
In addition tricks/tips to invoke an anabolic or anti-catabolic response in the absence of calories will be beneficial in a fast/semi-fast state. Intra-cellular hydration and leucine especially during a time of muscular blood flow (such as cardio) will be beneficial. In addition, IF leucine uptake is critical, as it is in the elderly and in fasted states, then the Isoleucine in BCAAs only compete w/ leucine for uptake while providing no benefit.
Transcripts related to ubiquitin-dependent protein catabolism and the ubiquitin cycle are higher after consumption of the lower protein diet and this suggests that inadequate dietary protein intake may initiate muscle protein degradation. Ubiquitin-dependent protein catabolism plays a primary role in skeletal muscle proteolysis during catabolic states [38] and [37] and this pathway was previously shown to be sensitive to leucine status [39]
Now this study gave inadequate protein intake for 12 days before looking for gene changes. So the results reflect more of a medium term adaptation versus the short-term adaptation of 18 hours, 24 hours, 36 hours... Still it provides a relevant understanding. Lessons include older adults are more susceptible to catabolism and have a lesser anabolic response. Thus they really need the higher protein intake and as previously indicated 3grams of leucine.
In addition tricks/tips to invoke an anabolic or anti-catabolic response in the absence of calories will be beneficial in a fast/semi-fast state. Intra-cellular hydration and leucine especially during a time of muscular blood flow (such as cardio) will be beneficial. In addition, IF leucine uptake is critical, as it is in the elderly and in fasted states, then the Isoleucine in BCAAs only compete w/ leucine for uptake while providing no benefit.
The skeletal muscle transcript profile reflects accommodative responses to inadequate protein intake in younger and older males, Anna E. Thalacker-Merce, The Journal of Nutritional Biochemistry Volume 21, Issue 11, November 2010, Pages 1076-1082
Abstract
Inadequate protein intake initiates adverse changes in skeletal muscle function and structure (i.e., an accommodative response). mRNA level changes due to short-term inadequate dietary protein might be an early indication of subsequent accommodation. The aims of this study were to assess the effects of dietary protein and the diet-by-age interaction on the skeletal muscle transcriptome.
Twelve younger (21–43 y) and 10 older (63–79 y) men completed three controlled feeding trials with protein intakes of 0.50 (LPro: lower protein), 0.75 (MPro: medium protein) and 1.00 g protein·kg body weight−1·day−1 (HPro: higher protein). A fasting state biopsy was taken on Day 12 of each trial. Global changes in transcript levels were assessed with Affymetrix genechips and expression patterns determined using self-organizing maps.
Nine hundred fifty-eight transcripts were differentially expressed (P<.05) by diet and 853 had a diet-by-age interaction (P<.05). The results for diet alone revealed that LPro was associated with up-regulation of transcripts related to ubiquitin-dependent protein catabolism and muscle contraction and LPro and MPro resulted in up-regulation of transcripts related to apoptosis and down-regulation of transcripts related to cell differentiation, muscle and organ development, extracellular space and responses to stimuli and stress. The diet-by-age effect on protein modification transcripts was consistent with the older males being less responsive to anabolic stimuli (lower protein synthesis at HPro) and more responsive to a catabolic state (protein breakdown at LPro). Changes in skeletal muscle mRNA levels in younger and older males to protein intake near or below the recommended dietary allowance are indicative of an early accommodative response.
1. Introduction
Dietary protein provides essential amino acids which are necessary for the synthesis of proteins and other biomolecules [1]. During an extended period of inadequate protein intake, structural and functional skeletal muscle proteins are catabolized [2] to obtain essential amino acids and a new steady state is developed with (i.e., accommodation [3]) or without (i.e., adaptation [4]) reduced physiological function. Accommodative responses to inadequate protein intake observed in older adults include decreased lean body and muscle masses, decreased muscle strength and function and a decreased immune response [5], [6] and [7]. These adverse physiological consequences may be especially problematic for older adults who already experience the involuntary decrease in skeletal muscle mass and strength that accompanies advancing age (sarcopenia). The Food and Nutrition Board of the Institute of Medicine has highlighted the need for more research to understand the events which precede the accommodative response to inadequate protein intake in skeletal muscle [8].
While microarray analysis does not determine mechanisms, it gives an unbiased global view of the molecular events that occur during various physiologic states and diseases. For example, skeletal muscle transcript levels respond to changes in energy [9], [10] and [11] and nutrient availability [12], [13] and [14] and mRNA level changes occur in human skeletal muscle [14] and rat liver [15] in response to inadequate protein intake. We have previously reported the global changes in skeletal muscle transcript levels that occur in older adults (67*7 years) following 1 week of inadequate protein intake [63% of the recommended dietary allowance (RDA) for protein] [14]. The changes in the transcript profile included up-regulation of transcripts related to contraction, movement and development; extracellular connective tissue; immune, inflammatory and stress responses and down-regulation of transcripts related to energy metabolism, protein synthesis and proliferation. However, that study could not determine if these were short-term responses to a decrease in protein intake or whether they were the first indication that the inadequate protein diet was stimulating an accommodative response in these older subjects. In addition, the study did not permit us to determine whether these changes are unique to older adults or could be generalized to younger adults. The present study was designed to assess these issues of age, dietary protein level and diet-by-age interactions on skeletal muscle transcript profiles. We hypothesize that the dietary protein-related transcript profile will reflect an accommodative response to inadequate protein intake and that age will blunt the beneficial effects of added protein intake on the muscle transcriptome.
...
...
Discussion
Previous studies have suggested that the current RDA for protein (0.8 g·kg−1·d−1) is inadequate to maintain protein homeostasis [5], [32], [33] and [31], fat-free mass and skeletal muscle cross-sectional area in older adults [7] and [34], while protein intake above the RDA may be needed to maintain lean body mass and type I muscle fiber cross sectional area in older women [5] and [6]. The results of our study reveal a molecular signature of a biological response to inadequate dietary protein intake that is consistent with this hypothesis. We believe that the observation that transcripts for “muscle and organ development” and “cell differentiation” increase when the protein intake is greater than the RDA (i.e., cluster C5 in Fig. 1), and previous phenotypic data [5], [6], [7], [32], [33], [34] and [31] suggest that there are positive responses to consuming protein in quantities greater than the RDA in skeletal muscle. Thus, our array data are consistent with previous reports that suggest a higher protein intake contributes to a healthier physiological profile [5], [6] and [35] and better muscle responses to resistance training [36]. The study duration was not, however, sufficient to directly link changes in the skeletal muscle transcript profile with previously reported phenotypes of adaptation and accommodation [5], [6], [7], [32], [33], [34] and [31].
The dietary protein-related changes we observed in skeletal muscle transcript levels appeared to be an early indication of an accommodative response to LPro and MPro diets in both the younger and older men. Many of these changes are consistent with phenotypes of accommodation observed in the study by Castaneda et al. where a 0.56 g protein·kg−1·d−1 diet was fed for 9 weeks [5] and [6]. Transcripts related to ubiquitin-dependent protein catabolism and the ubiquitin cycle are higher after consumption of the lower protein diet and this suggests that inadequate dietary protein intake may initiate muscle protein degradation. Ubiquitin-dependent protein catabolism plays a primary role in skeletal muscle proteolysis during catabolic states [38] and [37] and this pathway was previously shown to be sensitive to leucine status [39]. The IPA network developed from our data (Supplemental Figure 1) incorporates several ubiquitin-conjugating enzymes (UBE2N, UBE2D3, UBE2L3), UBA1 (the protein involved in the initial steps of protein degradation) and other molecules related to proteolysis (CFLAR AND SUMO3). We speculate that this response is initiated to maintain skeletal muscle remodeling and metabolism when dietary protein intake is limiting. However, these were up-regulated with the LPro diet in the older adults while they were down-regulated in the younger adults suggesting that youth provides some protection from the consequence of inadequate protein intake. Future research on age-dependent differences in the role of ubiquitin pathways in accommodative and adaptive responses to changes in dietary protein intake is warranted.
The suggestion that inadequate protein intake quickly induces an early molecular signature of the accommodative response in skeletal muscle is thematically consistent with the interpretation from our previous research. In that report, we showed that seven days of inadequate protein intake (0.5 g·kg−1·d−1, immediately after 7 d at 1.2 g·kg−1·d−1) induced alterations in the skeletal muscle transcript profile of older adults (67*7 y) [14]. However, interesting and potentially important differences exist between our current and previous studies [14]. First, the dietary protein-related changes in transcript levels were less robust in the current study (no differentially expressed transcripts met the FDR criterion, and only 21 transcripts were differentially expressed at P<.001) compared with our earlier study (85 transcripts that met the FDR criterion (P=.0006) and 49 transcripts that were significantly altered at P<.0001 [14]). Second, only 17 differentially expressed transcripts (at P<.05) were common between the two studies, and of these, only two were regulated in the same direction (Supplemental Table 4). The differences we observed between the transcript profile results from our two studies may be due to differences in study design. For example, the full cross-over design in the current study may have eliminated spurious findings due to inadequate controls in the pre/post analysis we used for our previous study. Additionally, the abrupt decrease in dietary protein intake (−0.7 g·kg−1·d−1) in the earlier study may have imposed a transient stress on the muscle that was reflected in the transcript profile. In contrast, the body may have had more time to recover from the shift to inadequate protein in the current study (i.e., 12-day feeding period and more modest shifts in dietary protein intake 0.25–0.5 g·kg−1·d−1). Collectively, our interpretation is that both studies are correct and that the differences reflect time-dependent changes that occur at the molecular level in the skeletal muscle in response to changes in dietary protein intake. This hypothesis must be tested directly in a longitudinal study with multiple sampling points and a longer feeding period.
Results from other microarray studies show that the muscle transcript profile changes with advancing age [41], [42], [43], [44], [45] and [40], and they reveal an up-regulation of transcripts involved with “RNA binding and splicing,” “hormone, growth factor, cytokine and signaling proteins,” and “protein degradation” as well as a down-regulation of transcripts related to “energy and mitochondrial metabolism” and “stress and inflammatory responses”. While these earlier studies did not control dietary intake and physical activity, the overall results are consistent with our current study and with the phenotypes of aging skeletal muscle (e.g., mitochondrial dysfunction; increased oxidative stress, apoptosis and RNA splicing; decreased energy metabolism). However, we also observed several age-related changes in the skeletal muscle transcript profile that are inconsistent with the physiological phenotype of sarcopenia, e.g., enrichment of transcripts for skeletal muscle and nervous system development. These transcript-level changes suggest aging muscle has initiated a compensatory mechanism to combat the age-related morphological and physiological changes in skeletal muscle.
The current study provides a unique opportunity to evaluate the interaction between age and dietary protein intake on muscle biology (Fig. 2, Supplemental Figure 1). Our data are consistent with reports of a blunted anabolic response of skeletal muscle to increasing amino acid intake in older adults [39], [47] and [46]. For example, the cluster analysis shows that transcripts related to protein metabolism were progressively up-regulated by increasing protein intake in the younger adults but were down-regulated in the older adults (clusters C0, C3 and C6, Fig. 2). Compared to the younger adults, when more exogenous amino acids are available (i.e., HPro) the older men had lower protein synthesis related transcript levels (C0) which may result in a decreased anabolic response to the high protein load. Additionally, the older men had higher transcript levels related to ubiquitin-dependent protein catabolism (C6) on the LPro, which suggests an increased catabolic state. The expression of transcripts related to protein modification (e.g., protein folding) corresponds to the effects on protein synthesis (down with HPro) and protein catabolism (up with LPro) in the older men. The important role of dietary protein in these processes is further demonstrated by identification of a network specific to post translational modification, protein degradation and protein synthesis for the transcripts with a diet-by-age interaction.
While the present study provides preliminary data and derived hypotheses for future mechanistic and phenotypic research, these data cannot be taken as causal and are merely descriptive. The transcript level is only one of the many regulatory steps that could be affected by changes in dietary protein intake; therefore, a longitudinal study, with sufficient time for measurable changes in the phenotype to occur and with multiple sampling points, is a necessary future step to directly relate the transcript-level phenotype to biochemical (e.g., total and phosphor-proteome), structural and functional alterations in skeletal muscle. Additional limitations of this study warrant further caution when evaluating these findings. The intensity of the expression changes we observed was modest and there are likely to be false positives in transcripts identified as differentially expressed given the P value of .05 that we used. However, several factors raise our confidence that our results are valid, i.e., RT-PCR analysis confirmed the direction of change in several transcripts, when multiple probe sets were available for a given transcript they were expressed and clustered similarly, family members and isoforms for a number of differentially expressed transcripts were frequently coregulated. Due to the limited availability of human muscle biopsy tissue we were unable to confirm whether molecular changes observed in a biological system are reflective of changes that occur in the protein or metabolic space. In addition, the RNA obtained from the biopsies was limited and we were therefore, forced to pool RNA to conduct follow-up RT-PCR validation analyses. Lastly, the differential results (discussed above), between the current and previous transcript profiling studies from our laboratory, presents a limitation to interpretation of these results. Future studies will allow us to resolve whether the differences between our two studies are due to acute but transient changes in the skeletal muscle transcript profile or whether they are a consequence of our earlier study design.
In summary, we report transcript level changes in younger and older males following just 2 weeks of inadequate and marginal protein intakes that are consistent with an early disruption of muscle biology. These molecular events may precede the changes in skeletal muscle function and structure that occur during accommodation to prolonged intake of diets containing inadequate protein. In older men, we also identify transcript level responses to changes in dietary protein intake that reflect a limit on anabolic responses and promotion of catabolic ones. If confirmed in future studies, this could explain why older adults are vulnerable to skeletal muscle atrophy with advancing age. In addition, our data are consistent with earlier reports that indicate physiologic benefits for older subjects that consume protein intakes that are moderately higher than the current RDA [5], [6], [7], [32], [33], [34] and [31]. The current study was not designed and cannot be used to determine the adequacy of the RDA for dietary protein intake for adults; however, if our transcript-level changes are confirmed and extended in necessary future studies, they could present a complementary assessment to other measures (e.g., nitrogen balance, isotope labeling) for addressing the adequacy of the RDA for dietary protein.
Abstract
Inadequate protein intake initiates adverse changes in skeletal muscle function and structure (i.e., an accommodative response). mRNA level changes due to short-term inadequate dietary protein might be an early indication of subsequent accommodation. The aims of this study were to assess the effects of dietary protein and the diet-by-age interaction on the skeletal muscle transcriptome.
Twelve younger (21–43 y) and 10 older (63–79 y) men completed three controlled feeding trials with protein intakes of 0.50 (LPro: lower protein), 0.75 (MPro: medium protein) and 1.00 g protein·kg body weight−1·day−1 (HPro: higher protein). A fasting state biopsy was taken on Day 12 of each trial. Global changes in transcript levels were assessed with Affymetrix genechips and expression patterns determined using self-organizing maps.
Nine hundred fifty-eight transcripts were differentially expressed (P<.05) by diet and 853 had a diet-by-age interaction (P<.05). The results for diet alone revealed that LPro was associated with up-regulation of transcripts related to ubiquitin-dependent protein catabolism and muscle contraction and LPro and MPro resulted in up-regulation of transcripts related to apoptosis and down-regulation of transcripts related to cell differentiation, muscle and organ development, extracellular space and responses to stimuli and stress. The diet-by-age effect on protein modification transcripts was consistent with the older males being less responsive to anabolic stimuli (lower protein synthesis at HPro) and more responsive to a catabolic state (protein breakdown at LPro). Changes in skeletal muscle mRNA levels in younger and older males to protein intake near or below the recommended dietary allowance are indicative of an early accommodative response.
1. Introduction
Dietary protein provides essential amino acids which are necessary for the synthesis of proteins and other biomolecules [1]. During an extended period of inadequate protein intake, structural and functional skeletal muscle proteins are catabolized [2] to obtain essential amino acids and a new steady state is developed with (i.e., accommodation [3]) or without (i.e., adaptation [4]) reduced physiological function. Accommodative responses to inadequate protein intake observed in older adults include decreased lean body and muscle masses, decreased muscle strength and function and a decreased immune response [5], [6] and [7]. These adverse physiological consequences may be especially problematic for older adults who already experience the involuntary decrease in skeletal muscle mass and strength that accompanies advancing age (sarcopenia). The Food and Nutrition Board of the Institute of Medicine has highlighted the need for more research to understand the events which precede the accommodative response to inadequate protein intake in skeletal muscle [8].
While microarray analysis does not determine mechanisms, it gives an unbiased global view of the molecular events that occur during various physiologic states and diseases. For example, skeletal muscle transcript levels respond to changes in energy [9], [10] and [11] and nutrient availability [12], [13] and [14] and mRNA level changes occur in human skeletal muscle [14] and rat liver [15] in response to inadequate protein intake. We have previously reported the global changes in skeletal muscle transcript levels that occur in older adults (67*7 years) following 1 week of inadequate protein intake [63% of the recommended dietary allowance (RDA) for protein] [14]. The changes in the transcript profile included up-regulation of transcripts related to contraction, movement and development; extracellular connective tissue; immune, inflammatory and stress responses and down-regulation of transcripts related to energy metabolism, protein synthesis and proliferation. However, that study could not determine if these were short-term responses to a decrease in protein intake or whether they were the first indication that the inadequate protein diet was stimulating an accommodative response in these older subjects. In addition, the study did not permit us to determine whether these changes are unique to older adults or could be generalized to younger adults. The present study was designed to assess these issues of age, dietary protein level and diet-by-age interactions on skeletal muscle transcript profiles. We hypothesize that the dietary protein-related transcript profile will reflect an accommodative response to inadequate protein intake and that age will blunt the beneficial effects of added protein intake on the muscle transcriptome.
...
...
Discussion
Previous studies have suggested that the current RDA for protein (0.8 g·kg−1·d−1) is inadequate to maintain protein homeostasis [5], [32], [33] and [31], fat-free mass and skeletal muscle cross-sectional area in older adults [7] and [34], while protein intake above the RDA may be needed to maintain lean body mass and type I muscle fiber cross sectional area in older women [5] and [6]. The results of our study reveal a molecular signature of a biological response to inadequate dietary protein intake that is consistent with this hypothesis. We believe that the observation that transcripts for “muscle and organ development” and “cell differentiation” increase when the protein intake is greater than the RDA (i.e., cluster C5 in Fig. 1), and previous phenotypic data [5], [6], [7], [32], [33], [34] and [31] suggest that there are positive responses to consuming protein in quantities greater than the RDA in skeletal muscle. Thus, our array data are consistent with previous reports that suggest a higher protein intake contributes to a healthier physiological profile [5], [6] and [35] and better muscle responses to resistance training [36]. The study duration was not, however, sufficient to directly link changes in the skeletal muscle transcript profile with previously reported phenotypes of adaptation and accommodation [5], [6], [7], [32], [33], [34] and [31].
The dietary protein-related changes we observed in skeletal muscle transcript levels appeared to be an early indication of an accommodative response to LPro and MPro diets in both the younger and older men. Many of these changes are consistent with phenotypes of accommodation observed in the study by Castaneda et al. where a 0.56 g protein·kg−1·d−1 diet was fed for 9 weeks [5] and [6]. Transcripts related to ubiquitin-dependent protein catabolism and the ubiquitin cycle are higher after consumption of the lower protein diet and this suggests that inadequate dietary protein intake may initiate muscle protein degradation. Ubiquitin-dependent protein catabolism plays a primary role in skeletal muscle proteolysis during catabolic states [38] and [37] and this pathway was previously shown to be sensitive to leucine status [39]. The IPA network developed from our data (Supplemental Figure 1) incorporates several ubiquitin-conjugating enzymes (UBE2N, UBE2D3, UBE2L3), UBA1 (the protein involved in the initial steps of protein degradation) and other molecules related to proteolysis (CFLAR AND SUMO3). We speculate that this response is initiated to maintain skeletal muscle remodeling and metabolism when dietary protein intake is limiting. However, these were up-regulated with the LPro diet in the older adults while they were down-regulated in the younger adults suggesting that youth provides some protection from the consequence of inadequate protein intake. Future research on age-dependent differences in the role of ubiquitin pathways in accommodative and adaptive responses to changes in dietary protein intake is warranted.
The suggestion that inadequate protein intake quickly induces an early molecular signature of the accommodative response in skeletal muscle is thematically consistent with the interpretation from our previous research. In that report, we showed that seven days of inadequate protein intake (0.5 g·kg−1·d−1, immediately after 7 d at 1.2 g·kg−1·d−1) induced alterations in the skeletal muscle transcript profile of older adults (67*7 y) [14]. However, interesting and potentially important differences exist between our current and previous studies [14]. First, the dietary protein-related changes in transcript levels were less robust in the current study (no differentially expressed transcripts met the FDR criterion, and only 21 transcripts were differentially expressed at P<.001) compared with our earlier study (85 transcripts that met the FDR criterion (P=.0006) and 49 transcripts that were significantly altered at P<.0001 [14]). Second, only 17 differentially expressed transcripts (at P<.05) were common between the two studies, and of these, only two were regulated in the same direction (Supplemental Table 4). The differences we observed between the transcript profile results from our two studies may be due to differences in study design. For example, the full cross-over design in the current study may have eliminated spurious findings due to inadequate controls in the pre/post analysis we used for our previous study. Additionally, the abrupt decrease in dietary protein intake (−0.7 g·kg−1·d−1) in the earlier study may have imposed a transient stress on the muscle that was reflected in the transcript profile. In contrast, the body may have had more time to recover from the shift to inadequate protein in the current study (i.e., 12-day feeding period and more modest shifts in dietary protein intake 0.25–0.5 g·kg−1·d−1). Collectively, our interpretation is that both studies are correct and that the differences reflect time-dependent changes that occur at the molecular level in the skeletal muscle in response to changes in dietary protein intake. This hypothesis must be tested directly in a longitudinal study with multiple sampling points and a longer feeding period.
Results from other microarray studies show that the muscle transcript profile changes with advancing age [41], [42], [43], [44], [45] and [40], and they reveal an up-regulation of transcripts involved with “RNA binding and splicing,” “hormone, growth factor, cytokine and signaling proteins,” and “protein degradation” as well as a down-regulation of transcripts related to “energy and mitochondrial metabolism” and “stress and inflammatory responses”. While these earlier studies did not control dietary intake and physical activity, the overall results are consistent with our current study and with the phenotypes of aging skeletal muscle (e.g., mitochondrial dysfunction; increased oxidative stress, apoptosis and RNA splicing; decreased energy metabolism). However, we also observed several age-related changes in the skeletal muscle transcript profile that are inconsistent with the physiological phenotype of sarcopenia, e.g., enrichment of transcripts for skeletal muscle and nervous system development. These transcript-level changes suggest aging muscle has initiated a compensatory mechanism to combat the age-related morphological and physiological changes in skeletal muscle.
The current study provides a unique opportunity to evaluate the interaction between age and dietary protein intake on muscle biology (Fig. 2, Supplemental Figure 1). Our data are consistent with reports of a blunted anabolic response of skeletal muscle to increasing amino acid intake in older adults [39], [47] and [46]. For example, the cluster analysis shows that transcripts related to protein metabolism were progressively up-regulated by increasing protein intake in the younger adults but were down-regulated in the older adults (clusters C0, C3 and C6, Fig. 2). Compared to the younger adults, when more exogenous amino acids are available (i.e., HPro) the older men had lower protein synthesis related transcript levels (C0) which may result in a decreased anabolic response to the high protein load. Additionally, the older men had higher transcript levels related to ubiquitin-dependent protein catabolism (C6) on the LPro, which suggests an increased catabolic state. The expression of transcripts related to protein modification (e.g., protein folding) corresponds to the effects on protein synthesis (down with HPro) and protein catabolism (up with LPro) in the older men. The important role of dietary protein in these processes is further demonstrated by identification of a network specific to post translational modification, protein degradation and protein synthesis for the transcripts with a diet-by-age interaction.
While the present study provides preliminary data and derived hypotheses for future mechanistic and phenotypic research, these data cannot be taken as causal and are merely descriptive. The transcript level is only one of the many regulatory steps that could be affected by changes in dietary protein intake; therefore, a longitudinal study, with sufficient time for measurable changes in the phenotype to occur and with multiple sampling points, is a necessary future step to directly relate the transcript-level phenotype to biochemical (e.g., total and phosphor-proteome), structural and functional alterations in skeletal muscle. Additional limitations of this study warrant further caution when evaluating these findings. The intensity of the expression changes we observed was modest and there are likely to be false positives in transcripts identified as differentially expressed given the P value of .05 that we used. However, several factors raise our confidence that our results are valid, i.e., RT-PCR analysis confirmed the direction of change in several transcripts, when multiple probe sets were available for a given transcript they were expressed and clustered similarly, family members and isoforms for a number of differentially expressed transcripts were frequently coregulated. Due to the limited availability of human muscle biopsy tissue we were unable to confirm whether molecular changes observed in a biological system are reflective of changes that occur in the protein or metabolic space. In addition, the RNA obtained from the biopsies was limited and we were therefore, forced to pool RNA to conduct follow-up RT-PCR validation analyses. Lastly, the differential results (discussed above), between the current and previous transcript profiling studies from our laboratory, presents a limitation to interpretation of these results. Future studies will allow us to resolve whether the differences between our two studies are due to acute but transient changes in the skeletal muscle transcript profile or whether they are a consequence of our earlier study design.
In summary, we report transcript level changes in younger and older males following just 2 weeks of inadequate and marginal protein intakes that are consistent with an early disruption of muscle biology. These molecular events may precede the changes in skeletal muscle function and structure that occur during accommodation to prolonged intake of diets containing inadequate protein. In older men, we also identify transcript level responses to changes in dietary protein intake that reflect a limit on anabolic responses and promotion of catabolic ones. If confirmed in future studies, this could explain why older adults are vulnerable to skeletal muscle atrophy with advancing age. In addition, our data are consistent with earlier reports that indicate physiologic benefits for older subjects that consume protein intakes that are moderately higher than the current RDA [5], [6], [7], [32], [33], [34] and [31]. The current study was not designed and cannot be used to determine the adequacy of the RDA for dietary protein intake for adults; however, if our transcript-level changes are confirmed and extended in necessary future studies, they could present a complementary assessment to other measures (e.g., nitrogen balance, isotope labeling) for addressing the adequacy of the RDA for dietary protein.
