A Higher-Protein, Energy Restriction Diet Containing 4 Servings of Fresh, Lean Beef per Day Does Not Negatively Influence Circulating miRNAs Associated with Cardiometabolic Disease Risk in Women with Overweight

This study examined the acute effects of 7-d energy restriction normal-protein (NP; ∼15% of daily intake as protein) compared with higher-protein (HP; ∼38% of daily intake as protein) diets varying in quantities of fresh, lean beef on circulating miRNA expression associated with cardiometabolic disease in 16 women with overweight (mean ± SD; age: 35 ± 8.7 y; body mass index: 28.5 ± 1.9 kg/m2). Fasting blood samples were collected at the end of each diet for miRNA expression, glucose, insulin, adiponectin, C-reactive protein (CRP), and IL-6. Of the 12 surveyed, 10 miRNAs (miR-320a-3p, miR-146a-5p, miR-150-5p, miR-423-5p, miR-122-5p, miR-223-3p, miR-199a-5p, miR-214-3p, miR-24-3p, and miR-126-3p) were detected. Several miRNAs were associated with fasting CRP (i.e., miR-150-5p, miR-24-3p, miR-423-5p; all P < 0.05). miR-423-5p was also associated with fasting glucose, IL-6, and homeostasis model assessment 2 %β cell function (all, P < 0.05). No differences in miRNA expression were identified between diets. These data suggest that fresh, lean beef in a short-term HP, energy restriction diet does not negatively influence circulating miRNAs associated with cardiometabolic disease in women. This trial was registered at clinicaltrials.gov as NCT02614729.


Introduction
In the United States, over 50% of adults have !1 chronic health condition with 30% experiencing multiple conditions, all of which reduce quality of life, life expectancy, and increase health care costs [1,2].Obesity is a leading cause of preventable death in the United States and is an independent risk factor for metabolic diseases including type 2 diabetes (T2D) and cardiovascular disease (CVD).Emerging regulators identified in the development and progression of chronic, metabolic diseases are a family of small (~22 nucleotides), noncoding RNA molecules known as microRNAs (miRNAs) that modulate gene expression at the posttranscriptional level [3].Triggered by local stimuli, including processes related to metabolic dysfunction (i.e., inflammation), miRNAs are released into circulation and regulate expression of genes in the same or in distal tissues including adipose tissue, pancreas, liver and muscle [4].Further, when comparing individuals who were metabolically healthy with those with a chronic condition (obesity, T2D), circulating miRNA profiles varied dependent on health status [5].Thus, aberrant expression of these posttranscriptional modifiers, as a result of excessive adiposity, contributes to sustained metabolic dysfunction [6][7][8].Although the relationship between chronic disease and miRNA expression continues to develop, the influence of dietary factors remains relatively unknown.
The most common dietary strategy promoted to combat obesity and reduce cardiometabolic disease risk includes energy restriction diets ranging from À500 to À750 kcal/d [9,10].
However, the ability to adhere to energy restriction diets over the longer term is challenging because of increased hunger and food cravings along with blunted satiety [10,11].Thus, 1 strategy to improve appetite control and satiety during energy restriction is through increasing dietary protein intake [12,13].Additional benefits of higher-protein (HP) diets (containing 27%-35% of daily energy as protein) include greater reductions in body weight and fat mass compared with standard protein diets (containing 16%-21% of energy as protein) as highlighted by a meta-analysis performed by Wycherley et al. [14].The majority of HP, energy restriction randomized controlled trials include animal-source protein-rich foods, particularly red meat, as part of the HP comparison [15,16].Observational evidence suggests that red meat consumption is highly correlated with increased risk of developing obesity, T2D, CVD, some cancers, and all-cause mortality [17].As such, the 2020-2025 Dietary Guidelines for Americans [17] emphasize healthy dietary patterns that include plant-based foods but limit the consumption of red and processed meats.However, a number of intervention-based trials evaluated within published meta-analyses [18,19] challenge these findings.Further, although fresh lean beef as part of a healthy pattern has been shown to be beneficial for CVD risk factors, total cholesterol, and LDL cholesterol [20], it is unclear as to whether these effects occur at the molecular level via changes in miRNA expression.
The purpose of this study was to identify whether an energy restriction, HP diet, containing ~4 servings of fresh, lean beef per day alters expression of circulating miRNAs selected based on their recorded signatures in obesity, T2D and CVD compared with an energy restriction, normal-protein (NP) diet containing ~1 serving of fresh, lean beef.

Experimental design
Secondary analyses were performed from an acute crossover design study in 16 women with overweight with no chronic conditions or diseases.The purpose of the original study was to examine the effects of NP compared with HP, energy restriction (À750 kcal/d) diets on appetite control, satiety, and ad libitum intake [21].Fasting blood samples were collected at the end of each dietary pattern for analysis of appetite and satiety hormones.In this analyses, fasting blood samples were analyzed for miRNA expression and markers of cardiometabolic disease risk.The original trial design is reported at clinicaltrials.govas NCT02614729.

Study participants
From January 2014 to May 2015, women with overweight were recruited from the Columbia, MO, area through advertisements, flyers, and e-mail listservs to participate in the study.Seventeen women signed the consent, began, and completed the study.Of those, 16 participants were included in this secondary analyses based on availability of fasting blood samples.In general, participants were adult women with overweight (mean AE SD; age: 35 AE 8.7 y; BMI: 28.5 AE 1.9 kg/m 2 ) with no chronic conditions or diseases.The majority (92%) were White and non-Hispanic/non-Latino.All participants were informed of the study purpose, procedures, and risks and signed the consent/assent forms.The study was approved by the University of Missouri Health Sciences institutional review board, and all procedures were followed in accordance with the ethical standards of the institutional review board.The participants received a stipend for completing all study procedures.

Diet interventions
For 7 d/diet, the participants were provided with isocaloric, energy-restricted (À750 kcal) diets as either NP or HP.The NP diet included 1240 AE 0 kcal/d (%daily energy: 15.5% protein; 55.5% carbohydrates; 31.2%fat), whereas the HP diet included 1280 AE 10 kcal/d (%daily energy: 38.8% protein; 39% carbohydrates; 23.2% fat).The sources of protein were similar across diets and consisted of fresh, lean beef (60% of total protein) as flank and top round steak and plant proteins (40% of total protein) as textured soy protein, tofu, and wheat gluten products.The NP diet contained ~1 serving/d of fresh, lean beef (4 ounces/d), whereas the HP diet contained ~4 servings/d of fresh, lean beef (15 ounces/d).

Fasting blood sampling and analyses
Blood samples (4 mL/sample) were collected after an overnight fast at the end of each 7-d period.Collection, processing, and storage methods are published elsewhere [22].

Data and statistical analyses
Power analyses were performed from data generated by a study evaluating dietary intervention influence on circulating miRNA expression [23].A sample size of 9 was suggested to provide 80% power to detect meaningful differences in expression from the mean differential of 3.98 (fold change) and SD of difference of 3.56, an effect size of 1.12.β-Cell function, insulin resistance and sensitivity were examined via homeostasis model assessment (HOMA) 2-insulin resistance, HOMA2 β-cell function (%), and HOMA2-insulin sensitivity (%) calculations (https:// www.dtu.ox.ac.uk/homacalculator/). Summary statistics were generated and normality assessment of the data via Shapiro-Wilk test was performed before statistical testing.Outliers, identified as data points falling outside 1.5 times the interquartile range below Q1 or above Q3 were removed from the data set.All miRNA data were log-transformed (log 2 ) for statistical analyses.Paired-sample t tests were performed to evaluate the impact of the quantity of beef consumption during energy restriction on miRNA expression and markers of acute inflammation and glycemic control.If paired samples did not cross the C T in both NP and HP diets, these samples were excluded from the analyses.
Pearson correlational analyses were performed to identify associations between the expression of circulating miRNAs and fasting cardiometabolic biomarkers.Each statistical test was performed using P 0.05 as criterion for statistical significance.Statistical analyses were performed in IBM SPSS Statistics (version: 28.0.0.0).

Results
A total of 10 of the 12 a priori miRNAs were detected following both diets as shown in Figure 1.No differences in the expression of miRNAs surveyed were observed between the NP and HP diets.Correlational analyses performed to identify miRNA-cardiometabolic marker associations are reported in Table 1.A number of miRNAs were positively associated with fasting C-reactive protein (i.e., miR-150-5p, miR-24-3p, miR-423-5p; all P < 0.05).Further, miR-423-5p was inversely associated with fasting glucose and positively associated with IL-6 and HOMA2-β cell function (all P < 0.05).

Discussion
Despite the varying protein quantity between these 7-d energy restriction diets (difference of: 76 g protein/d), no differences in miRNA expression were detected in women with overweight with no chronic conditions or diseases.These data suggest that the acute consumption of 4 servings of fresh, lean beef in a HP, energy restriction diet does not negatively influence miRNAs identified as potential mediators of cardiometabolic disease risk.
A limited number of intervention-based studies exist examining whether dietary factors alter CVD risk via circulating miRNA expression [24].Of those that involve energy restriction, these studies vary in duration and dietary approach to evaluate the impact of glycemic index and load [25], macronutrient compositions [26], exercise [27][28][29], and meal replacements [30] on miRNA expression.Parr et al. [27] compared 16-wk diets varying in dairy protein and carbohydrates within an exercise-induced energy restriction on circulating miRNAs similar to those in this study.All interventions led to significant weight loss, which was accompanied by an increased expression of miR-223-3p, a suggested biomarker for obesity.We did not detect differences in miR-223-3p in this study for a number of potential reasons.The 7-d intervention design in this study limited our ability to assess weight loss.In addition, we included protein-rich foods from beef, whereas Parr et al. [27] included dairy; the differences in protein source may have also contributed to variable findings.

TABLE 1
Pearson correlations between the relative expression of circulating miRNA and fasting cardiometabolic markers analyzed after the consumption of the normal-protein (NP) and higher-protein (HP) diets.A recent systematic review of dietary impact on miRNA regulation [24] highlights that specific dietary factors (e.g., unsaturated fatty acids, plant-based foods) can regulate expression of the same miRNA differently.For example, in our previous study, we found that miR-15b-5p expression was higher after 7 d of consuming an energy balance diet containing fresh, lean beef compared with a diet void of fresh, lean beef [22].The lack of difference in the panel of miRNAs surveyed in this study compared with other studies could be explained by the varying dietary factors, including protein quality and energy balance, across studies.
There are limitations to highlight, most notably that not all the miRNAs surveyed consistently crossed the C T , which resulted in a variable sample size across the analysis.We achieved !30% detection of miRNAs surveyed in the total sample, which is above the 20% cutoff incorporated in most studies [26].Regardless, we want to mention several potential reasons for this occurrence.First, a single miRNA can be differentially expressed across different dietary patterns and across the magnitude of response to the intervention [24].In addition, miRNA expression can vary between individuals based on whether those individuals are sensitive and responsive to the dietary interventions [26,27].Finally, although sample collection can also impact the ability to detect miRNA expression in plasma, the samples collected in this study were stored and processed appropriately to detect circulating miRNAs.Additionally, baseline samples were not collected in this study, and thus, no preanalyses/postanalyses were available.The intervention was also short in duration (7 d) and is thus not generalizable to long-term changes.Although adequately powered, the sample size (N ¼ 16) was relatively small with a fairly homogenous population of healthy women with overweight.Therefore, long-term randomized controlled trials with premeasurements and postmeasurements are needed to elucidate the impact of higher protein consumption, varying in protein quality and/or energy status, on a larger panel of miRNAs involved in cardiometabolic disease risk and their associated gene targets.
Collectively, our findings suggest that including fresh, lean beef in a short-term higher protein, healthy dietary pattern during energy restriction does not negatively influence circulating miRNAs associated with cardiometabolic disease development.

Funding
The Beef Checkoff supplied the funds to complete the study but was not involved in the design, implementation, analyses or interpretation of the data.

FIGURE 1 .
FIGURE 1. Dot plots of circulating miRNA for comparison between the normal-protein (NP) and higher-protein (HP) diets via paired-sample t tests.Values are means AE SD.

TABLE 1 (
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