Katsanos, Christos S.

Lipoprotein Lipase (LPL) hydrolyzes plasma triglycerides into free fatty acids in the circulation for use and/or storage in tissues (i.e., adipose tissue, skeletal muscle). Impaired LPL activity (LPLa) is associated with unfavorable health outcomes, such as elevated plasma triglyceride concentrations. We sought to determine how plasma LPLa relates to body composition measurements in individuals with varying degrees of adiposity. Eighteen subjects (ages 19 to 45 years) with body mass index (BMI) between 18 and 45 kg/m2 had their body composition measured by dual-energy X-ray absorptiometry (DEXA) and insulin sensitivity estimated from an oral glucose tolerance test by using the Matsuda Index (MI). Subjects ingested a bolus of 5g fat (i.e., whipping cream, 0.2 g fat/kg fat free mass), followed by small boluses of fat over the course of a 3-hour experimental period. This experimental protocol allows for the induction of a postprandial state without perturbation of the plasma triglyceride or insulin concentrations. LPLa was measured in the presence of increased plasma insulin induced by intravenous insulin infusion during the last 30 mins of the 3-hour experimental protocol. Insulin was infused at a rate of 0.5 mU/kg/min for subjects with MI-determined insulin sensitivity < 4 and 1 mU/kg/min for those with MI-determined insulin sensitivity > 4, in order to produce comparable levels of plasma insulin concentrations across subjects. To release endothelial-bound LPL, subjects received an injection of intravenous heparin (75 UI/kg) at the end of the plasma insulin infusion. Plasma samples were collected 10 minutes after the heparin infusion and analyzed for LPL concentration (LPLc) and LPLa using commercially available ELISA kits. Person’s correlation was used to evaluate relationships between several body composition parameters and LPLc as well as LPLa. LPLc was not correlated (P >0.05) with BMI (r = 0.03), total lean body mass (r = -0.20), total body fat (r = -0.02), or android fat (r = - 0.08). However, LPLa was positively correlated (P <0.05) with BMI (r = 0.49) and total lean body mass (r = 0.58), as well as total body fat (r = 0.54, P = 0.02) and android fat (r = 0.58, P = 0.01). In conclusion, increased body fat, including android fat, are associated with increased intravascular LPLa in the presence of insulin. Greater insulin-stimulated intravascular LPLa can enhance the release of triglyceride-associated free fatty acids and their uptake by tissues, resulting in insulin resistance as documented in individuals with increased android fat distribution. American Diabetes Association Grant #7-12-CT-40. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

AbstractObjectiveThis 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.MethodsThe 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.ResultsAlong 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.ConclusionsThese findings indicate that lower protein synthesis observed in muscle of humans with obesity may result from diminished muscle IGF1 gene expression.

Abstract 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.

We recently discovered persistent downregulation of the H + ‐ATP synthase catalytic subunit (β‐F1‐ATPase) in skeletal muscle of obese individuals, which was linked to comorbid hyperlipidemia. β‐F1‐ATPase expression is primarily regulated post‐transcription, i.e. miRNAs, but the precise mechanism impeding expression are unclear. We tested the hypothesis that hyperlipidemia reduces β‐F1‐ATPase translational efficiency and is mediated by miR‐127‐5p, a miRNA known to control β‐F1‐ATPase translation. Biopsies isolated from the vastus lateralis of lean (BMI蠄25; n=7) and obese (BMI蠅30; n=6) subjects were evaluated for mRNA/miRNA expression via qRT‐PCR, protein by Western blot, and translation efficiency was calculated as the mRNA/protein ratio. Our results confirmed β‐F1‐ATPase translation was lower in obese individuals (P<0.01; 0.25±0.05%) compared to healthy weight controls (0.59±0.09%), and correlated with miR‐127‐5p expression (P<0.01; r=‐0.67). Bisulfite sequencing analysis of the miR‐127‐5p promoter revealed two hypomethylated sites in obese individuals, which may sustain an upregulation. Moreover, we found miR‐127‐5p present in muscle‐derived blood exosomes suggesting putative involvement in intracellular cross‐talk. In primary myotubes from a lean volunteer, prolonged fatty acid exposure increased miR‐127‐5p expression. Free Fatty Acid (mEq/L) Unsaturated/ Saturated mRNA (2 ‐ DD Ct ) Protein (AU) Translation (%) CTRL 0.347 ‐ 1.70±0.113 0.216±0.062 0.130±0.040 Lo Fat 0.185 0.55 1.60±0.094 0.202±0.038 0.123±0.008 Hi Fat 0.251 2.29 1.35±0.133* 0.095±0.006* 0.073±0.010 Hi Fat 0.269 0.54 1.66±0.145 0.093±0.003* 0.050±0.004* * P <0.05; One‐way ANOVA, Tukey9s posttest Inhibiting miR‐127‐5p with locked nucleic acids dose‐dependently improved β‐F1‐ATPase translation. Inhibitor (nM) mRNA (2 ‐ DD Ct ) Protein (AU) Translation (%) 0 1.84±0.314 0.22±0.062 0.647±0.120 10 1.93±0.387 1.24±0.135* 0.500±0.148 50 2.00±0.498 1.24±0.242** 1.483±0.233* 100 1.76±0.207 1.54±0.086* 0.892±0.096 * P <0.05; ** P <0.01; *** P <0.001; 1‐way ANOVA, Tukey9s posttest Our results provide evidence implicating hyperlipidemia‐induced miR‐127‐5p expression in suppressing β‐F1‐ATPase translation in human obesity. Funding from NIH/NIDDK grant DK094062 to CSK

Whether obesity influences content and functional capacity of skeletal muscle mitochondria (MITO) remains unresolved. In this study, biopsies of vastus lateralis from lean and obese human subjects were used to isolate subsarcolemmal (SS) and intermyofibrillar (IMF) MITO. IMF MITO were liberated using a protease. The SS and IMF were pelleted separately, using differential centrifugation. Skeletal muscle from obese subjects gave higher MITO protein yield, per g wet muscle vs. skeletal muscle from lean subjects in the SS but not in the IMF. There was a trend for higher isolated yields of citrate synthase (CS) activity, a marker for MITO content, in the muscle of obese subjects vs. lean subjects in SS, but not in IMF MITO. O 2 consumption ( J O ) and ATP production rates measured with NAD‐linked substrates (malate+pyruvate+glutamate), were not different in obese vs. lean in either SS or IMF MITO expressed either per mg MITO protein or per unit CS activity. The similarities in MITO function observed in SS and IMF from both lean and obese human muscle, suggests comparable functional capacity per unit MITO, regardless of the location within the myocyte, or whether the MITO reside in skeletal muscle from lean or obese subjects. However, higher total yield of both protein and CS activities in the SS MITO from the muscle of obese subjects, point to the possibility for greater content of SS MITO in muscle of obese individuals. This conclusion must be qualified; these results may be due to a greater fractional yield of MITO during the isolation procedure of skeletal muscle from obese subjects.

The amino acid enrichment in blood (E b ) is often used in clinically relevant stable isotope studies to calculate muscle protein fractional synthesis rates (FSR), but it underestimates the FSR compared to methods using intracellular enrichment in muscle (E i ). We compared the FSR calculated with intravenous infusion of the novel tracer L‐[2,3,3,4,5,5,5,6,6,6‐ 2 H 10 ]leucine ([ 2 H 10 ]leu) to infusion of the traditional tracer L‐[ 13 C 6 ]phenylalanine ([ 13 C 6 ]phe) using E b and E i . Because [ 2 H 10 ]leu is intracellularly metabolized to [ 2 H 9 ]leu prior to reentry into the blood, we hypothesize that using [ 2 H 10 ]leu will minimize the discrepancy between FSR values calculated using E b or E i compared to [ 13 C 6 ]phe‐calculated FSR values using the corresponding E b or E i . To test our hypothesis, six healthy subjects received simultaneous infusions of the two stable‐isotope tracers following standard protocols. The [ 2 H 9 ]leu‐calculated FSR (%h ‐1 ) was 0.08±0.01 based on E b and 0.18±0.01 based on E i . The corresponding [ 13 C 6 ]phe‐calculated FSR values were 0.06±0.01 and 0.25±0.02, respectively. Moreover, the difference in the [ 2 H 9 ]leu‐calculated FSR values using E i vs E b was significantly less ( P <0.01; 0.10±0.01) compared to the difference in the corresponding [ 13 C 6 ]phe‐calculated FSR values (0.19±0.02). We conclude that, compared to [ 13 C 6 ]phe, the [ 2 H 10 ]leu tracer offers a more accurate approach to calculating FSR when E b is sought as an alternative to the E i. Grant Funding Source : Supported by NIH/NIDDK Grant# R01 DK094062; NCATS Grant# UL1 TR000135

Multiple conditions are linked to perturbed skeletal muscle protein (SMP) metabolism, including excess adiposity and insulin resistance. Here we delineate the precise roles of body fat and insulin sensitivity on SMP metabolism, and test the hypothesis that hyperaminoacidemia can eliminate the influence of these factors. Six healthy individuals underwent a DEXA scan to determine percent body fat (%BF; 16.3‐29.4%) and an oral glucose tolerance test (OGTT) to evaluate insulin sensitivity via Matsuda index (MI; 1.21‐12.0). Subjects received a primed, continuous infusion of L‐[2,3,3,4,5,5,5,6,6,6‐ 2 H 10 ]leucine to determine SMP fractional synthesis rate (FSR) in biopsies from the vastus lateralis . Myostatin (MSTN) mRNA, a marker of muscle wasting, was quantified in similar biopsies, and MSTN activation was assessed in plasma as the percent of active over latent protein. To evaluate the effect of hyperaminoacidemia, samples were collected before and during infusions of high amino acids (AA). In our subjects, %BF was not correlated with MI ( P =0.11). The %BF was only associated with MSTN mRNA ( P =0.02; r=0.81). However, MI was a key determinant in FSR ( P =0.02; r=0.83) and MSTN activation ( P <0.01; r=‐0.94). AA infusion eliminated all discrepancies in FSR and MSTN quantitations. Thus, SMP metabolism is more strongly linked to insulin sensitivity than body fat, but the influence is attenuated with hyperaminoacidemia. Grant Funding Source : Supported by NIH/NIDDK Grant# R01 DK094062; NCATS Grant# UL1 TR000135