Sphingosine-1-phosphate analog FTY720 reverses obesity but not age-induced anabolic resistance to muscle contraction.

Sarcopenia, the age-associated loss of skeletal muscle mass and function, is coupled with declines in physical functioning leading to subsequent higher rates of disability, frailty, morbidity, and mortality. Aging and obesity independently contribute to muscle atrophy that is assumed to be a result of the activation of mutual physiological pathways. Understanding mechanisms contributing to the induction of skeletal muscle atrophy with aging and obesity is important for determining targets that may have pivotal roles in muscle loss in these conditions. We find that aging and obesity equally induce an anabolic resistance to acute skeletal muscle contraction as observed with decreases in anabolic signaling activation after contraction. Furthermore, treatment with the sphingosine-1-phosphate analog FTY720 for 4 wk increased lean mass and strength, and the anabolic signaling response to contraction was improved in obese but not older animals. To determine the role of chronic inflammation and different fatty acids on anabolic resistance in skeletal muscle cells, we overexpressed IKKß with and without exposure to saturated fatty acid (SFA; palmitic acid), polyunsaturated fatty acid (eicosapentaenoic acid), and monounsaturated fatty acid (oleic acid). We found that IKKß overexpression increased inflammation markers in muscle cells, and this chronic inflammation exacerbated anabolic resistance in response to SFA. Pretreatment with FTY720 reversed the inflammatory effects of palmitic acid in the muscle cells. Taken together, these data demonstrate chronic inflammation can induce anabolic resistance, SFA aggravates these effects, and FTY720 can reverse this by decreasing ceramide accumulation in skeletal muscle.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

Differential expression of CCR8 in tumors versus normal tissue allows specific depletion of tumor-infiltrating T regulatory cells by GS-1811, a novel Fc-optimized anti-CCR8 antibody.

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

Effects of Low Doses of L-Carnitine Tartrate and Lipid Multi-Particulate Formulated Creatine Monohydrate on Muscle Protein Synthesis in Myoblasts and Bioavailability in Humans and Rodents.

The primary objective of this study was to investigate the potential synergy between low doses of L-carnitine tartrate and creatine monohydrate to induce muscle protein synthesis and anabolic pathway activation in primary human myoblasts. In addition, the effects of Lipid multi-particulates (LMP) formulation on creatine stability and bioavailability were assessed in rodents and healthy human subjects. When used individually, L-carnitine tartrate at 50 µM and creatine monohydrate at 0.5 µM did not affect myoblast protein synthesis and signaling. However, when combined, they led to a significant increase in protein synthesis. Increased AKT and RPS6 phosphorylation were observed with 50 µM L-carnitine tartrate 5 µM creatine in combination in primary human myoblasts. When Wistar rats were administered creatine with LMP formulation at either 21 or 51 mg/kg, bioavailability was increased by 27% based on the increase in the area under the curve (AUC) at a 51 mg/kg dose compared to without LMP formulation. Tmax and Cmax were unchanged. Finally, in human subjects, a combination of LMP formulated L-carnitine at 500 mg (from L-carnitine tartrate) with LMP formulated creatine at 100, 200, or 500 mg revealed a significant and dose-dependent increase in plasma creatine concentrations. Serum total L-carnitine levels rose in a similar manner in the three combinations. These results suggest that a combination of low doses of L-carnitine tartrate and creatine monohydrate may lead to a significant and synergistic enhancement of muscle protein synthesis and activation of anabolic signaling. In addition, the LMP formulation of creatine improved its bioavailability. L-carnitine at 500 mg and LMP-formulated creatine at 200 or 500 mg may be useful for future clinical trials to evaluate the effects on muscle protein synthesis.

Circulating MicroRNA Are Predictive of Aging and Acute Adaptive Response to Resistance Exercise in Men.

Circulating microRNA (c-miRNA) have the potential to function as novel noninvasive markers of the underlying physiological state of skeletal muscle. This investigation sought to determine the influence of aging on c-miRNA expression at rest and following resistance exercise in male volunteers (Young: n = 9; Older: n = 9). Primary findings were that fasting c-miRNA expression profiles were significantly predictive of aging, with miR-19b-3p, miR-206, and miR-486 distinguishing between age groups. Following resistance exercise, principal component analysis revealed a divergent response in expression of 10 c-miRNA, where expression profiles were upregulated in younger and downregulated in older participants. Using Ingenuity Pathway Analysis to test c-miRNA-to-mRNA interactions in skeletal muscle, it was found that response of c-miRNA to exercise was indicative of an anabolic response in younger but not older participants. These findings were corroborated with a positive association observed with the phosphorylation status of p-AktSer473 and p-S6K1Thr389 and expression of miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-26b-5p, miR-143-3p, and miR-195-5p. These important findings provide compelling evidence that dysregulation of c-miRNA expression with aging may not only serve as a predictive marker, but also reflect underlying molecular mechanisms resulting in age-associated declines in skeletal muscle mass, increased fat mass, and "anabolic resistance."

Effects of Potassium Bicarbonate Supplements on Circulating microRNA Expression.

Several studies suggest that neutralizing acid load in the diet with alkali had favorable effects on intermediate markers of musculoskeletal health. We examined whether alkali supplementation with potassium bicarbonate [(KHCO3); 81 mmol/d; n = 12] vs placebo (n = 12) for 84 days altered serum microRNAs, potential biomarkers associated with innumerable biological processes including bone and muscle metabolism. Serum microRNAs, urinary net acid excretion (UNAE), urinary N-telopeptide (UNTX), urinary calcium (UCa), urinary nitrogen (UN), glomerular filtration rate, serum procollagen type 1 amino-terminal propeptide (P1NP), serum insulin-like growth factor-1 (IGF-1), and its serum binding protein IGFBP3 were measured at baseline and day 84. Baseline characteristics and measurements were similar in the two treatment groups. Eighty-four-day changes in UNAE differed by group (KHCO3, -47 ± 9 mmol; placebo, -5 ± 5 mmol; P < 0.01). KHCO3 significantly reduced UNTX, UCa, and serum P1NP but did not affect UN, serum IGF-1, or IGFBP3 levels compared with placebo over 84 days. Fold change in serum circulating microRNA (c-miR)-133b differed significantly by group (KHCO3, 2.26 ± 0.85; placebo, -1.23 ± 0.69; P < 0.01); there was a similar trend in c-miR-21-5p. Fold changes in c-miR-133b and c-miR-21-5p were inversely associated with changes in UNAE and UNTX; fold change in c-miR-21-5p was inversely associated with change in UCa, with a similar trend with c-miR-133b. In summary, reducing renal acid load with KHCO3 was associated with increased expressions of c-miR-133b and c-miR-21-5p. Furthermore, increases in c-miRNA-133b and c-miR-21-5p were inversely associated with bone resorption markers UNTX and UCa consistent with potential beneficial effects on bone in older adults. However, the broader significance of c-miRNAs as musculoskeletal biomarkers is still under investigation, and larger studies are needed to verify these preliminary results.

Calorie Restricted High Protein Diets Downregulate Lipogenesis and Lower Intrahepatic Triglyceride Concentrations in Male Rats.

The purpose of this investigation was to assess the influence of calorie restriction (CR) alone, higher-protein/lower-carbohydrate intake alone, and combined CR higher-protein/lower-carbohydrate intake on glucose homeostasis, hepatic de novo lipogenesis (DNL), and intrahepatic triglycerides. Twelve-week old male Sprague Dawley rats consumed ad libitum (AL) or CR (40% restriction), adequate (10%), or high (32%) protein (PRO) milk-based diets for 16 weeks. Metabolic profiles were assessed in serum, and intrahepatic triglyceride concentrations and molecular markers of de novo lipogenesis were determined in liver. Independent of calorie intake, 32% PRO tended to result in lower homeostatic model assessment of insulin resistance (HOMA-IR) values compared to 10% PRO, while insulin and homeostatic model assessment of ß-cell function (HOMA-ß) values were lower in CR than AL, regardless of protein intake. Intrahepatic triglyceride concentrations were 27.4 ± 4.5 and 11.7 ± 4.5 µmol·g(-1) lower (p < 0.05) in CR and 32% PRO compared to AL and 10% PRO, respectively. Gene expression of fatty acid synthase (FASN), stearoyl-CoA destaurase-1 (SCD1) and pyruvate dehydrogenase kinase, isozyme 4 (PDK4) were 45% ± 1%, 23% ± 1%, and 57% ± 1% lower (p < 0.05), respectively, in CR than AL, regardless of protein intake. Total protein of FASN and SCD were 50% ± 1% and 26% ± 1% lower (p < 0.05) in 32% PRO compared to 10% PRO, independent of calorie intake. Results from this investigation provide evidence that the metabolic health benefits associated with CR-specifically reduction in intrahepatic triglyceride content-may be enhanced by consuming a higher-protein/lower-carbohydrate diet.

Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression.

Short-term (5-10 days) calorie restriction (CR) downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1), however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR). Twelve-week old male Sprague Dawley rats consumed ad libitum (AL) or calorie restricted (CR; 40%) adequate (10%, AIN-93M) or high (32%) protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05) in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6, and p70S6K were lower (P < 0.05) in CR vs. AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36). Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.