Very Low Calorie Ketogenic Diet: What Effects on Lipid Metabolism?

PURPOSE OF REVIEW: This review aims to critically examine how VLCKD affects plasma lipoprotein, lipid and cholesterol metabolism. Cardiovascular disease is a worldwide health problem affecting millions of people and leading to high rates of mortality and morbidity. There is a well-established association between cardiovascular disease and circulating cholesterol. Various dietary recommendations are currently available for the management of dyslipidemia. RECENT FINDINGS: The very low-calorie ketogenic diet (VLCKD) is becoming increasingly popular as a treatment option for several pathological conditions, including dyslipidemia. In addition to being low in calories, the VLCKD's main feature is its unique calorie distribution, emphasizing a reduction in carbohydrate consumption in favor of fat as the primary calorie source. Lowering calorie intake through a VLCKD can reduce the endogenous production of cholesterol. However, if the foods consumed are from animal sources, dietary cholesterol intake may increase due to the higher fat content of animal products. When combined, these dietary practices may have opposing effects on plasma cholesterol levels. Studies investigating the impact of VLCKD on plasma cholesterol and low-density lipoprotein cholesterol levels report contradictory findings. While some studies found an increase in low-density lipoprotein cholesterol levels, others showed a decrease in total cholesterol and low-density lipoprotein cholesterol, along with an increase in high-density lipoprotein cholesterol.

Impaired Suppression of Plasma Lipid Extraction and its Partitioning Away from Muscle by Insulin in Humans with Obesity.

Context: Humans with obesity and insulin resistance exhibit lipid accumulation in skeletal muscle, but the underlying biological mechanisms responsible for the accumulation of lipid in the muscle of these individuals remain unknown. Objective: We investigated how plasma insulin modulates the extraction of circulating triglycerides (TGs) and non-esterified fatty acids (NEFAs) from dietary and endogenous sources in the muscle of lean, insulin-sensitive humans (Lean-IS) and contrasted these responses to those in humans with obesity and insulin resistance (Obese-IR). Methods: The studies were performed in a postprandial state associated with steady-state plasma TG concentrations. The arterio-venous blood sampling technique was employed to determine the extraction of circulating lipids across the forearm muscle before and after insulin infusion. We distinguished kinetics of TGs and NEFAs from dietary sources across muscle from those from endogenous sources by incorporating stable isotope-labeled triolein in ingested fat. Results: Plasma insulin rapidly suppressed the extraction of plasma TGs from endogenous, but not dietary, sources in the Lean-IS, but same response was absent in the Obese-IR. Furthermore, in the muscle of Lean-IS, plasma insulin decreased the extraction of circulating NEFAs from both dietary and endogenous sources, but in Obese-IR subjects this response was absent for NEFAs from dietary sources. Conclusions: Partitioning of circulating lipids away from the skeletal muscle when plasma insulin increases, such as during the postprandial period, is impaired in humans with obesity and insulin resistance. Trial Registration: ClinicalTrials.gov ( NCT01860911 ).

Obesity and Obesity-Related Thyroid Dysfunction: Any Potential Role for the Very Low-Calorie Ketogenic Diet (VLCKD)?

PURPOSE OF REVIEW: This review aims to explore in-depth the different aspects of the association between very low-calorie ketogenic diet (VLCKD), obesity and obesity-related thyroid dysfunction. RECENT FINDINGS: The VLCKD, proposed as a non-pharmacological strategy for the management of certain chronic diseases, is becoming increasingly popular worldwide. Initially used to treat epilepsy, it has been shown to be effective in controlling body weight gain and addressing various pathophysiological conditions. Research has shown that a low-calorie, high-fat diet can affect thyroid hormone levels. Weight loss can also influence thyroid hormone levels. Studies have suggested that long-term use of VLCKD for refractory epilepsy may be related to the development of hypothyroidism, with an effect seen in various populations. In particular, women with obesity following VLCKD tend to have reduced T3 levels. We propose further research to unravel the underlying mechanisms linking VLCKD to obesity and obesity-related thyroid dysfunction.

Lower muscle protein synthesis in humans with obesity concurrent with lower expression of muscle IGF1 splice variants.

OBJECTIVE: This study tested the hypothesis that expression of insulin-like growth factor 1 (IGF-1) protein and mRNA splice variants is lower in skeletal muscle of humans with obesity who have a lower mixed-muscle protein fractional synthesis rate (MMP-FSR) when compared with individuals without obesity. METHODS: The study included nine participants with obesity (OB, mean [SD],  BMI = 35 [3] kg/m2 , MMP-FSR = 0.06%/h [0.02%/h]) and nine participants without obesity (W-OB, BMI = 24 [3] kg/m2 , MMP-FSR = 0.08%/h [0.02%/h]; for both BMI and MMP-FSR p < 0.05). MMP-FSR and mitochondrial protein FSR were measured following an overnight fast. RESULTS: Along with lower MMP-FSR, OB participants displayed lower mitochondrial protein FSR (p = 0.03) compared with W-OB participants. Expression of IGF-1 (p = 0.04) and IGF-1 receptor (p < 0.01) proteins was lower in muscle of OB participants. In addition, OB participants had lower (p < 0.05) mRNA expression of IGF1 variants Eb and Ec. This study demonstrates that lower protein synthesis in muscle of humans with obesity occurs concurrently with lower expression of muscle IGF-1 and IGF-1 receptor proteins, as well as lower mRNA expression of the IGF1 splice variants. CONCLUSIONS: These findings indicate that lower protein synthesis observed in muscle of humans with obesity may result from diminished muscle IGF1 gene expression.

Muscle fiber phenotype: a culprit of abnormal metabolism and function in skeletal muscle of humans with obesity.

The proportion of the different types of fibers in a given skeletal muscle contributes to its overall metabolic and functional characteristics. Greater proportion of type I muscle fibers is associated with favorable oxidative metabolism and function of the muscle. Humans with obesity have a lower proportion of type I muscle fibers. We discuss how lower proportion of type I fibers in skeletal muscle of humans with obesity may explain metabolic and functional abnormalities reported in these individuals. These include lower muscle glucose disposal rate, mitochondrial content, protein synthesis, and quality/contractile function, as well as increased risk for heart disease, lower levels of physical activity, and propensity for weight gain/resistance to weight loss. We delineate future research directions and the need to examine hybrid muscle fiber populations, which are indicative of a transitory state of fiber phenotype within skeletal muscle. We also describe methodologies for precisely characterizing muscle fibers and gene expression at the single muscle fiber level to enhance our understanding of the regulation of muscle fiber phenotype in obesity. By contextualizing research in the field of muscle fiber type in obesity, we lay a foundation for future advancements and pave the way for translation of this knowledge to address impaired metabolism and function in obesity.

Increased Expression of Syncytin-1 in Skeletal Muscle of Humans With Increased Body Mass Index.

Obesity negatively impacts skeletal muscle protein metabolism, and also impairs skeletal muscle maintenance and regeneration. We analyzed muscle biopsy samples from humans with increased body mass index (BMI) (i.e. > 30 kg/m2) and controls (i.e., BMI < 25 kg/m2) for expression of syncytin-1, a fusogenic protein regulating skeletal muscle regeneration. When compared to controls, humans with increased BMI and concomitant reduction in muscle protein synthesis had higher expression of syncytin-1 in skeletal muscle (p < 0.05). Across human subjects, muscle protein synthesis correlated inversely (r = -0.51; p = 0.03) with syncytin-1 expression in muscle. Using a C2C12 cell line we found that expression of syncytin-A (i.e, corresponding protein in murine tissue) is increased by insulin, and that this response is impaired in the presence of fatty acids, whose metabolism is altered within the metabolic environment induced by increased BMI. In C2C12 cells, the response of the protein 4E-BP1, which signals increase in protein synthesis in muscle, resembled that of syncytin-A. These findings provide novel insights into the expression of syncytin-1 in skeletal muscle of humans with increased BMI, as well as its basic regulation by insulin and fatty acids in muscle. The findings signify the need for further research into the regulation of syncytin-1 in skeletal muscle of humans with increased BMI, as well as its biological implications for altering muscle protein metabolism and regeneration.

(Dys)regulation of Protein Metabolism in Skeletal Muscle of Humans With Obesity.

Studies investigating the proteome of skeletal muscle present clear evidence that protein metabolism is altered in muscle of humans with obesity. Moreover, muscle quality (i.e., strength per unit of muscle mass) appears lower in humans with obesity. However, relevant evidence to date describing the protein turnover, a process that determines content and quality of protein, in muscle of humans with obesity is quite inconsistent. This is due, at least in part, to heterogeneity in protein turnover in skeletal muscle of humans with obesity. Although not always evident at the mixed-muscle protein level, the rate of synthesis is generally lower in myofibrillar and mitochondrial proteins in muscle of humans with obesity. Moreover, alterations in the synthesis of protein in muscle of humans with obesity are manifested more readily under conditions that stimulate protein synthesis in muscle, including the fed state, increased plasma amino acid availability to muscle, and exercise. Current evidence supports various biological mechanisms explaining impairments in protein synthesis in muscle of humans with obesity, but this evidence is rather limited and needs to be reproduced under more defined experimental conditions. Expanding our current knowledge with direct measurements of protein breakdown in muscle, and more importantly of protein turnover on a protein by protein basis, will enhance our understanding of how obesity modifies the proteome (content and quality) in muscle of humans with obesity.

Regulation of Voluntary Physical Activity Behavior: A Review of Evidence Involving Dopaminergic Pathways in the Brain.

Physical activity leads to well-established health benefits. Current efforts to enhance physical activity have targeted mainly socioeconomic factors. However, despite these efforts, only a small number of adults engage in regular physical activity to the point of meeting current recommendations. Evidence collected in rodent models and humans establish a strong central nervous system component that regulates physical activity behavior. In particular, dopaminergic pathways in the central nervous system are among the best-characterized biological mechanisms to date with respect to regulating reward, motivation, and habit formation, which are critical for establishing regular physical activity. Herein, we discuss evidence for a role of brain dopamine in the regulation of voluntary physical activity behavior based on selective breeding and pharmacological studies in rodents, as well as genetic studies in both rodents and humans. While these studies establish a role of dopamine and associated mechanisms in the brain in the regulation of voluntary physical activity behavior, there is clearly need for more research on the underlying biology involved in motivation for physical activity and the formation of a physical activity habit. Such knowledge at the basic science level may ultimately be translated into better strategies to enhance physical activity levels within the society.

Lack of Increase in Muscle Mitochondrial Protein Synthesis During the Course of Aerobic Exercise and Its Recovery in the Fasting State Irrespective of Obesity.

Acute aerobic exercise induces skeletal muscle mitochondrial gene expression, which in turn can increase muscle mitochondrial protein synthesis. In this regard, the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), is a master regulator of mitochondrial biogenesis, and thus mitochondrial protein synthesis. However, PGC-1α expression is impaired in muscle of humans with obesity in response to acute aerobic exercise. Therefore, we sought to determine whether muscle mitochondrial protein synthesis is also impaired under the same conditions in humans with obesity. To this end, we measured mitochondrial and mixed-muscle protein synthesis in skeletal muscle of untrained subjects with (body fat: 34.7 ± 2.3%) and without (body fat: 25.3 ± 3.3%) obesity in a basal period and during a continuous period that included a 45 min cycling exercise (performed at an intensity corresponding to 65% of heart rate reserve) and a 3-h post-exercise recovery. Exercise increased PGC-1α mRNA expression in muscle of subjects without obesity, but not in subjects with obesity. However, muscle mitochondrial protein synthesis did not increase in either subject group. Similarly, mixed-muscle protein synthesis did not increase in either group. Concentrations of plasma amino acids decreased post-exercise in the subjects without obesity, but not in the subjects with obesity. We conclude that neither mitochondrial nor mixed-muscle protein synthesis increase in muscle of humans during the course of a session of aerobic exercise and its recovery period in the fasting state irrespective of obesity. Trial Registration: The study has been registered within ClinicalTrials.gov (NCT01824173).

Subpopulation-specific differences in skeletal muscle mitochondria in humans with obesity: insights from studies employing acute nutritional and exercise stimuli.

Mitochondria from skeletal muscle of humans with obesity often display alterations with respect to their morphology, proteome, biogenesis, and function. These changes in muscle mitochondria are considered to contribute to metabolic abnormalities observed in humans with obesity. Most of the evidence describing alterations in muscle mitochondria in humans with obesity, however, lacks reference to a specific subcellular location. This is despite data over the years showing differences in the morphology and function of subsarcolemmal (found near the plasma membrane) and intermyofibrillar (nested between the myofibrils) mitochondria in skeletal muscle. Recent studies reveal that impairments in mitochondrial function in obesity with respect to the subcellular location of the mitochondria in muscle are more readily evident following exposure of the skeletal muscle to physiological stimuli. In this review, we highlight the need to understand skeletal muscle mitochondria metabolism in obesity in a subpopulation-specific manner and in the presence of physiological stimuli that modify mitochondrial function in vivo. Experimental approaches employed under these conditions will allow for more precise characterization of impairments in skeletal muscle mitochondria and their implications in inducing metabolic dysfunction in human obesity.

Mitochondrial ATP synthase ß-subunit production rate and ATP synthase specific activity are reduced in skeletal muscle of humans with obesity.

NEW FINDINGS: What is the central question of this study? Humans with obesity have lower ATP synthesis in muscle along with lower content of the ß-subunit of the ATP synthase (ß-F1-ATPase), the catalytic component of the ATP synthase. Does lower synthesis rate of ß-F1-ATPase in muscle contribute to these responses in humans with obesity? What is the main finding and its importance? Humans with obesity have a lower synthesis rate of ß-F1 -ATPase and ATP synthase specific activity in muscle. These findings indicate that reduced production of subunits forming the ATP synthase in muscle may contribute to impaired generation of ATP in obesity. ABSTRACT: The content of the ß-subunit of the ATP synthase (ß-F1 -ATPase), which forms the catalytic site of the enzyme ATP synthase, is reduced in muscle of obese humans, along with a reduced capacity for ATP synthesis. We studied 18 young (37 ± 8 years) subjects of which nine were lean (BMI = 23 ± 2 kg m-2 ) and nine were obese (BMI = 34 ± 3 kg m-2 ) to determine the fractional synthesis rate (FSR) and gene expression of ß-F1 -ATPase, as well as the specific activity of the ATP synthase. FSR of ß-F1 -ATPase was determined using a combination of isotope tracer infusion and muscle biopsies. Gene expression of ß-F1 -ATPase and specific activity of the ATP synthase were determined in the muscle biopsies. When compared to lean, obese subjects had lower muscle ß-F1 -ATPase FSR (0.10 ± 0.05 vs. 0.06 ± 0.03% h-1 ; P < 0.05) and protein expression (P < 0.05), but not mRNA expression (P > 0.05). Across subjects, abundance of ß-F1 -ATPase correlated with the FSR of ß-F1 -ATPase (P < 0.05). The specific activity of muscle ATP synthase was lower in obese compared to lean subjects (0.035 ± 0.004 vs. 0.042 ± 0.007 arbitrary units; P < 0.05), but this difference was not significant after the activity of the ATP synthase was adjusted to the ß-F1 -ATPase content (P > 0.05). Obesity impairs the synthesis of ß-F1 -ATPase in muscle at the translational level, reducing the content of ß-F1 -ATPase in parallel with reduced capacity for ATP generation via the ATP synthase complex.

Adenosine Triphosphate Production of Muscle Mitochondria after Acute Exercise in Lean and Obese Humans.

INTRODUCTION: Current evidence indicates mitochondrial dysfunction in humans with obesity. Acute exercise appears to enhance mitochondrial function in the muscle of nonobese humans, but its effects on mitochondrial function in muscle of humans with obesity are not known. We sought to determine whether acute aerobic exercise stimulates mitochondrial function in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in humans with obesity. METHODS: We assessed maximal adenosine triphosphate production rate (MAPR) and citrate synthase (CS) activity in isolated SS and IMF mitochondria from subjects with body mass index < 27 kg·m (median age, 25 yr; interquartile range, 22-39 yr) and subjects with body mass index > 32 kg·m (median age, 29 yr; interquartile range, 20-39 yr) before and 3 h after a 45-min cycling exercise at an intensity corresponding to 65% HR reserve. The SS and IMF mitochondria were isolated from muscle biopsies using differential centrifugation. Maximal adenosine triphosphate production rate and CS activities were determined using luciferase-based and spectrophotometric enzyme-based assays, respectively. RESULTS: Exercise increased MAPR in IMF mitochondria in both nonobese subjects and subjects with obesity (P < 0.05), but CS-specific activity did not change in either group (P > 0.05). Exercise increased MAPR supported by complex II in SS mitochondria, in both groups (P < 0.05), but MAPR supported by complex I or palmitate did not increase by exercise in the subjects with obesity (P > 0.05). Citrate synthase-specific activity increased in SS mitochondria in response to exercise only in nonobese subjects (P < 0.05). CONCLUSIONS: In nonobese humans, acute aerobic exercise increases MAPR in both SS and IMF mitochondria. In humans with obesity, the exercise increases MAPR in IMF mitochondria, but this response is less evident in SS mitochondria.

Obesity modifies the stoichiometry of mitochondrial proteins in a way that is distinct to the subcellular localization of the mitochondria in skeletal muscle.

BACKGROUND: Skeletal muscle mitochondrial content and function appear to be altered in obesity. Mitochondria in muscle are found in well-defined regions within cells, and they are arranged in a way that form distinct subpopulations of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We sought to investigate differences in the proteomes of SS and IMF mitochondria between lean subjects and subjects with obesity. METHODS: We performed comparative proteomic analyses on SS and IMF mitochondria isolated from muscle samples obtained from lean subjects and subjects with obesity. Mitochondria were isolated using differential centrifugation, and proteins were subjected to label-free quantitative tandem mass spectrometry analyses. Collected data were evaluated for abundance of mitochondrial proteins using spectral counting. The Reactome pathway database was used to determine metabolic pathways that are altered in obesity. RESULTS: Among proteins, 73 and 41 proteins showed different (mostly lower) expression in subjects with obesity in the SS and IMF mitochondria, respectively (false discovery rate-adjusted P ≤ 0.05). We specifically found an increase in proteins forming the tricarboxylic acid cycle and electron transport chain (ETC) complex II, but a decrease in proteins forming protein complexes I and III of the ETC and adenosine triphosphate (ATP) synthase in subjects with obesity in the IMF, but not SS, mitochondria. Obesity was associated with differential effects on metabolic pathways linked to protein translation in the SS mitochondria and ATP formation in the IMF mitochondria. CONCLUSIONS: Obesity alters the expression of mitochondrial proteins regulating key metabolic processes in skeletal muscle, and these effects are distinct to mitochondrial subpopulations located in different regions of the muscle fibers. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01824173).

Lower Fasted-State but Greater Increase in Muscle Protein Synthesis in Response to Elevated Plasma Amino Acids in Obesity.

OBJECTIVE: Obesity alters protein metabolism in skeletal muscle, but consistent evidence is lacking. This study compared muscle protein synthesis in adults with obesity and in lean controls in the fasted state and during an amino acid infusion. METHODS: Ten subjects with obesity (age: 36 ± 3 years; BMI: 34 ± 1 kg/m2 ) and ten controls (age: 35 ± 3 years; BMI: 23 ± 1 kg/m2 ) received an infusion of L-[2,3,3,4,5,5,5,6,6,6-2 H10 ]leucine (0.15 µmol/kg fat-free mass/min) to measure muscle protein synthesis after an overnight fast and during amino acid infusion. RESULTS: Despite greater muscle mammalian target of rapamycin phosphorylation (P ≤ 0.05), fasted-state mixed-muscle and mitochondrial protein synthesis were lower in subjects with obesity (P ≤ 0.05). However, the change in mixed-muscle protein synthesis during the amino acid infusion was 2.7-fold greater in subjects with obesity (P ≤ 0.05), accompanied by a greater change in S6 kinase-1 phosphorylation (P ≤ 0.05). The change in mitochondrial protein synthesis did not differ between groups (P > 0.05). CONCLUSIONS: Adults with obesity have reduced muscle protein synthesis in the fasted state, but this response is compensated for by a greater change in overall muscle protein synthesis during amino acid infusion.

Plasma Amino Acids Stimulate Uncoupled Respiration of Muscle Subsarcolemmal Mitochondria in Lean but Not Obese Humans.

Context: Obesity is associated with mitochondrial dysfunction in skeletal muscle. Increasing the plasma amino acid (AA) concentrations stimulates mitochondrial adenosine triphosphate (ATP) production in lean individuals. Objective: To determine whether acute elevation in plasma AAs enhances muscle mitochondrial respiration and ATP production in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in obese adults. Design: Assessment of SS and IMF mitochondrial function during saline (i.e., control) and AA infusions. Participants: Eligible participants were healthy lean (body mass index, <25 kg/m2; age, 37 ± 3 years; n = 10) and obese (body mass index >30 kg/m2; age 35 ± 3 years; n = 11) subjects. Intervention: Single trial of saline infusion followed by AA infusion. SS and IMF mitochondria were isolated from muscle biopsies collected at the end of the saline and AA infusions. Main Outcomes: Mitochondrial respiration and ATP production. Results: AA infusion increased adenosine 5'-diphosphate (ADP)-stimulated respiration and ATP production rates of SS mitochondria in the lean (P < 0.05), but not obese, subjects. Furthermore, AA infusion increased the uncoupled (i.e., non-ADP-stimulated) respiration of SS mitochondria in the lean subjects only (P < 0.05). AA infusion had no effect on any of these parameters in IMF mitochondria in either lean or obese subjects (P > 0.05). Conclusions: Increasing the plasma AA concentrations enhances the capacity for respiration and ATP production of muscle SS, but not IMF, mitochondria in lean individuals, in parallel with increases in uncoupled respiration. However, neither of these parameters increases in muscle SS or IMF mitochondria in obese individuals.

Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.

Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P < 0.01) whole body phenylalanine rate of appearance (µmol·kg-1·min-1), indicating suppression of muscle proteolysis, in both the control (1.02 ± 0.04 vs 0.76 ± 0.04) and the BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA.

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase ß Subunit.

Our previous studies show reduced abundance of the ß-subunit of mitochondrial H+-ATP synthase (ß-F1-ATPase) in skeletal muscle of obese individuals. The ß-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing ß-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of ß-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of ß-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of ß-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of ß-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with ß-F1-ATPase protein translation efficiency in humans (r = - 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased ß-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired ß-F1-ATPase translation as an important consequence of obesity.

Subsarcolemmal mitochondria isolated with the proteolytic enzyme nagarse exhibit greater protein specific activities and functional coupling.

Skeletal muscle mitochondria are arranged as a reticulum. Insight into the functional characteristics of such structure is achieved by viewing the network as consisting of "subsarcolemmal" (SS) and "intermyofibrillar" (IMF) regions. During the decades, most, but not all, published studies have reported higher (sometimes over 2-fold) enzyme and enzyme-pathway protein-specific activities in IMF compared to SS mitochondria. We tested the hypothesis that non-mitochondrial protein contamination might account for much of the apparently lower specific activities of isolated SS mitochondria. Mouse gastrocnemii (n = 6) were suspended in isolation medium, minced, and homogenized according to procedures typically used to isolate SS mitochondria. However, the supernatant fraction, collected after the first slow-speed (800×g) centrifugation, was divided equally: one sample was exposed to nagarse (MITO+), while the other was not (MITO-). Nagarse treatment reduced total protein yield by 25%, while it increased protein-specific respiration rates (nmol O2 min-1 mg-1), by 38% under "resting" (state 4) and by 84% under maximal (state 3) conditions. Nagarse therefore increased the respiratory control ratio (state 3/state 4) by 30%. In addition, the ADP/O ratio was increased by 9% and the activity of citrate synthase (U/mg) was 49% higher. Mass spectrometry analysis indicated that the MITO+ preparation contained less contamination from non-mitochondrial proteins. We conclude that nagarse treatment of SS mitochondria removes not only non-mitochondrial proteins but also the protein of damaged mitochondria, improves indices of functional integrity, and the resulting protein-specific activities.

Combined effects of aerobic exercise and l-arginine ingestion on blood pressure in normotensive postmenopausal women: A crossover study.

After menopause the incidence of cardiovascular diseases increases in women. A decrease in nitric oxide (NO) bioavailability has been pointed out to play a major role in this phenomenon. Since it is believed that l-arginine administration could improve NO bioavailability, the aim of this study was to examine the effects of acute l-arginine administration associated with aerobic exercise on blood pressure (BP), redox state and inflammatory biomarkers in normotensive postmenopausal women (NPW). Sixteen volunteers (57±6yr) were subjected to four experimental sessions (crossover design): arginine+exercise (A-E); arginine (ARG); exercise+placebo (EXE); control (CON). Each session was initiated with either 9g of l-arginine ingestion (ARG or A-E days), placebo (EXE day), or nothing (CON day). The participants performed 30min of aerobic exercise (A-E and EXE days) or sitting rest (CON and ARG days). Blood samples were collected before each session and 45min after the intervention. Office BP and ambulatory blood pressure monitoring (ABPM) were evaluated. NO/cGMP pathway, redox state and inflammatory biomarkers were measured. Systolic BP decreased during the 24-hour in A-E and EXE sessions. However, diastolic BP reduced only in A-E session. No changes were found in the biomarkers concentrations. In conclusion, the association was effective in lowering diastolic BP in NPW. Additionally, physical exercise alone promoted a long lasting effect on systolic BP measured by ABPM in this population, although this beneficial effect was not associated with changes in the cardio-inflammatory biomarkers. Possibly, other factors such as neural influences could be mediating this effect.