L6 Myocytes Research Articles

Antidiabetic, anti-inflammatory, antioxidant, and cytotoxicity potentials of green-synthesized zinc oxide nanoparticles using the aqueous extract of Helichrysum cymosum

The current research involved the synthesis of zinc oxide nanoparticles (ZnO-NPs) using an aqueous extract of Helichrysum cymosum shoots, and subsequent characterization via different analytical methods, such as UV–Vis spectroscopy, Scanning electron microscope (SEM), Energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), Transmission electron microscope (TEM), and zeta potential. The biological effects of the ZnO-NPs were then tested against C3A hepatocyte cells and L6 myocyte cell lines via series of analysis, including cytotoxicity, antioxidant, anti-inflammatory, and antidiabetic effect via enzymatic inhibition. The UV–Vis analysis showed a maximum absorption spectrum at 360, and the TEM analysis reveals a spherical and hexagonal structures, with an average dimension of 28.05–58.3 nm, and the XRD reveals a crystalline hexagonal structure. The zeta potential evaluation indicated that the ZnO-NPs are relatively stable at − 20 mV, and the FTIR analysis identified some important functional group associated with phenolics, carboxylic acid, and amides that are responsible for reducing and stabilizing the ZnO-NPs. The synthesized ZnO-NPs demonstrated cytotoxic effects on the cell lines at higher concentrations (125 µg/mL and 250 µg/mL), complicating the interpretation of the results of the inflammatory and antioxidant assays. However, there was a significant (p < 0.05) increase in the inhibitions of pancreatic lipase, alpha-glucosidase, and alpha-amylase, indicating beneficial antidiabetic effects.

N-3 and n-6 Polyunsaturated Fatty Acids Modulate Macrophage-Myocyte Inflammatory Crosstalk and Improve Myocyte Insulin Sensitivity.

In obesity, circulating saturated fatty acids (SFAs) and inflammatory cytokines interfere with skeletal muscle insulin signaling, leading to whole body insulin resistance. Further, obese skeletal muscle is characterized by macrophage infiltration and polarization to the inflammatory M1 phenotype, which is central to the development of local inflammation and insulin resistance. While skeletal muscle-infiltrated macrophage-myocyte crosstalk is exacerbated by SFA, the effects of other fatty acids, such as n-3 and n-6 polyunsaturated fatty acids (PUFAs), are less studied. Thus, the objective of this study was to determine the effects of long-chain n-3 and n-6 PUFAs on macrophage M1 polarization and subsequent effects on myocyte inflammation and metabolic function compared to SFA. Using an in vitro model recapitulating obese skeletal muscle cells, differentiated L6 myocytes were cultured for 24 h with RAW 264.7 macrophage-conditioned media (MCM), followed by insulin stimulation (100 nM, 20 min). MCM was generated by pre-treating macrophages for 24 h with 100 μM palmitic acid (16:0, PA-control), arachidonic acid (20:4n-6, AA), or docosahexaenoic acid (22:6n-3, DHA). Next, macrophage cultures were stimulated with a physiological dose (10 ng/mL) of lipopolysaccharide for an additional 12 h to mimic in vivo obese endotoxin levels. Compared to PA, both AA and DHA reduced mRNA expression and/or secreted protein levels of markers for M1 (TNFα, IL-6, iNOS; p < 0.05) and increased those for M2 (IL-10, TGF-β; p < 0.05) macrophage polarization. In turn, AA- and DHA-derived MCM reduced L6 myocyte-secreted cytokines (TNFα, IL-6; p < 0.05) and chemokines (MCP-1, MIP-1β; p < 0.05). Only AA-derived MCM increased L6-myocyte phosphorylation of Akt (p < 0.05), yet this was inconsistent with improved insulin signaling, as only DHA-derived MCM improved L6 myocyte glucose uptake (p < 0.05). In conclusion, dietary n-3 and n-6 PUFAs may be a useful strategy to modulate macrophage-myocyte inflammatory crosstalk and improve myocyte insulin sensitivity in obesity.

Alteration of zeta potential and cell viability in rat-derived L6 skeletal muscle cells and H9c2 cardiomyocytes: A study with submicron polystyrene particles.

Microand nanoplastics pollution can cause substantial damage to ecosystems. Since scientists have focused mainly on their impact on aquatic environments, less attention has been paid to the accumulation of polymer particles in terrestrial organisms. We checked if submicron (<5 mm) polystyrene (PS) particles, which can accumulate in living organisms, lead to changes in the physicochemical properties of mammalian cell membranes. The influence of submicron PS particles on the properties of rat-derived L6 myocytes and H9c2 cardiomyocytes was analyzed. Non-functionalized and amine-functionalized PS particles of 100 nm and 200 nm in diameter were used. The MTT assay was performed to evaluate the viability of the polymers-treated cells. The effect of short (6 h) and prolonged (48 h) incubation with different concentrations of PS particles on the cell's zeta (ζ) potential was examined with the electrophoretic light scattering technique (ELS). Polystyrene particles' physicochemical characteristics (size and stability) were performed using dynamic light scattering (DLS) and electrophoretic light scattering methods. The results show that submicron PS particles affect cell viability and cause changes in the physiochemical parameters of rat cell membranes. Differences were observed depending on the origin of the cells. We observed doseand time-dependent alterations in the studied parameters after submicron PS particle incubation in L6 myotubes and H9c2 cardiomyocytes. The size and modification of PS particle surfaces determine the extent to which they affect the analyzed properties of rat cardiomyocytes and myocytes membranes.

Nitrite lowers the oxygen cost of ATP supply in cultured skeletal muscle cells by stimulating the rate of glycolytic ATP synthesis.

Dietary nitrate lowers the oxygen cost of human exercise. This effect has been suggested to result from stimulation of coupling efficiency of skeletal muscle oxidative phosphorylation by reduced nitrate derivatives. In this paper, we report the acute effects of sodium nitrite on the bioenergetic behaviour of cultured rat (L6) myocytes. At odds with improved efficiency of mitochondrial ATP synthesis, extracellular flux analysis reveals that a ½-hour exposure to NaNO2 (0.1–5 μM) does not affect mitochondrial coupling efficiency in static myoblasts or in spontaneously contracting myotubes. Unexpectedly, NaNO2 stimulates the rate of glycolytic ATP production in both myoblasts and myotubes. Increased ATP supply through glycolysis does not emerge at the expense of oxidative phosphorylation, which means that NaNO2 acutely increases the rate of overall myocellular ATP synthesis, significantly so in myoblasts and tending towards significance in contractile myotubes. Notably, NaNO2 exposure shifts myocytes to a more glycolytic bioenergetic phenotype. Mitochondrial oxygen consumption does not decrease after NaNO2 exposure, and non-mitochondrial respiration tends to drop. When total ATP synthesis rates are expressed in relation to total cellular oxygen consumption rates, it thus transpires that NaNO2 lowers the oxygen cost of ATP supply in cultured L6 myocytes.

Sirt3 negatively regulates Glut4 in skeletal muscle insulin resistance in old male offspring rats fed with maternal high fat diet

High fat (HF) diet intake during pregnancy is linked to insulin resistance (IR) in offspring, but the molecular mechanism still needs to be clarified. In the present study, pregnant rats were fed either HF diet (45% energy from fat) or normal control (CON) diet (10% energy from fat) during gestation from day 5–21. Experiments were performed in 20 months old male offspring rats. The results showed that the fasting insulin levels of old male offspring in HF increased compared with CON. However, the fasting glucose levels was unchanged. Besides, the glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were impaired, and the AUC of GTT in HF and CON was 44.62±5.12, 33.58±3.98, with significant differences (P<.05). Furthermore, the AUC of ITT in HF and CON was 16.62±2.07 and 12.44±2.28, which showed an obvious difference (P<.05). Additionally, Sirt3 transcript and protein expressions were found up-regulated in both of the HF old male offspring skeletal muscle (1.42-fold) (P<.05) and palmitic acid (PA) treated L6 myotubes (1.32-fold) (P<.05), accompanied with increase in nicotinamide adenine dinucleotide (NAD+) in HF old male offspring skeletal muscle (P<.05). What's more, Glut4 transcript and protein expressions were found decreased significantly in both of the HF old male offspring skeletal muscle (0.45-fold) (P<.01) and PA treated L6 myotubes (0.49-fold) (P<.01). Transfection of Sirt3 gene in differentiated L6 myocytes indicated that Sirt3 negatively regulated Glut4 in L6 cells. However, AcK-103 protein, an indicator of acetylation, was found decreased in HF old male offspring skeletal muscle and L6 myotubes as a result of up-regulation of Sirt3 (P<.05). Thus, our findings suggest that HF diet intake during pregnancy induced IR in the skeletal muscle of old male offspring rats, probably occurred through Sirt3-Glut4 retrograde regulation, providing novel information concerning the molecular mechanism of IR in old male offspring rats.

Disruption of neuromedin B receptor improves mitochondrial oxidative phosphorylation capacity in gastrocnemius muscle of female mice.

Neuromedin B (NB), a bombesin-like peptide, exerts its specific actions by binding to the neuromedin B receptor (NBR), a G protein-coupled receptor. Female NBR-knockout (NBR-KO) mice exhibit resistance to diet-induced obesity, without hyperphagia, suggesting possible increase in energy expenditure. Skeletal muscle (SM) is crucial for whole body energy homeostasis, however, the presence of NB-NBR signaling and its effects in SM are unknown. Here, we show that male and female wild type express Nmbr and Nmb mRNA in SM, with higher levels in females. Female NBR-KO gastrocnemius showed increased Myh7 mRNA level, which characterizes type I fibers (oxidative profile). Their permeabilized gastrocnemius fibers, studied by high-resolution respirometry, exhibited higher consumption of O2 coupled to ATP synthesis and unaltered uncoupled respiration. NBR-KO gastrocnemius had higher protein levels of ATP-synthase and Nduf9 mRNA, corresponding to mitochondrial complex I subunit. NBR-KO gastrocnemius exhibited slight increase in mitochondria number, increased thickness of Z line at electron microscopy, and unaltered mitochondrial dynamics markers. Therefore, in the females' gastrocnemius, a predominantly glycolytic SM, the NBR absence promotes changes that favor mitochondrial oxidative phosphorylation capacity. In addition, in L6 myocytes, NB treatment (5 μg/mL/16 h) promoted lower O2 consumption coupled to ATP synthesis, suggesting direct action at SM cells. Altogether, the study reinforces the hypothesis that inhibition of NB-NBR signaling enhances the capacity for oxidative phosphorylation of white SM, encouraging future studies to elucidate their contribution on other types of SM and whole body energy expenditure, which may lead to a new target to drug development for obesity treatment.NEW & NOTEWORTHY This study describes neuromedin B (NB) and NB receptor as new regulators of skeletal muscle mitochondrial function. The white skeletal muscle mitochondrial oxidative phosphorylation capacity was increased by NB receptor genetic disruption in female mice. These findings may contribute to the resistance to diet-induced obesity, previously found in these mice, which requires future studies. Thus, investigations are necessary to clarify if blockade of NB receptor may be an approach to develop drugs to combat obesity.

The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells.

Simple SummaryPro-androgenic substances such as testosterone are often used to treat muscle- or bone-related disorders. Their interactions with the classical androgen receptor, however, can trigger a number of undesirable effects. It would therefore be of great benefit if the positive androgenic effects could be obtained by circumventing the classical androgen receptor. ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance. Using in silico methods, we identified and verified the extracellular localization of its androgen binding site and designed small peptides that fit in it that do not interact with the AR. All peptides were found to be pro-androgenic; they stimulate mineralization in osteoblastic cells and myogenesis in myoblasts. Thus, these peptides might serve as testosterone surrogates in the treatment of osteogenic or myogenic disorders.ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.

The involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1 activity in C2C12 and L6 myocytes

ObjectiveIGF-I and branched-chain amino acids have been reported to promote muscle hypertrophy via the stimulation of protein synthesis. Sestrin2, the function of which is regulated by leucine, has been reported to attenuate the activity of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) that stimulates protein synthesis. The objective of this study was to examine whether IGF-I modulates Sestrin2 abundance and to clarify the involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1. DesignC2C12 and L6 myocytes were stimulated by leucine (1 mM) with or without pretreatment with IGF-I (100 ng/mL). Phosphorylation of p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), both of which are targets of the mTORC1, was examined by western blotting. Effects of Sestrin2 small interfering RNA (siRNA) on the actions of leucine and IGF-I were examined. Sestrin2 mRNA and protein levels were also determined after Sestrin2 siRNA. ResultsLeucine increased the phosphorylation of S6K and 4E-BP1 in a dose-dependent manner. Pretreatment with IGF-I for 5 h further increased the stimulatory effect of leucine on the phosphorylation of S6K and 4E-BP1 in C2C12 cells. IGF-I increased Sestrin2 protein and messenger RNA levels. Sestrin2 siRNA increased or tended to increase basal phosphorylation of 4E-BP1 and decreased the leucine-induced phosphorylation in C2C12 and L6 cells, in particular after IGF-I treatment, suggesting the involvement of Sestrin2 in the action of leucine and IGF-I. The net increase in leucine-induced 4E-BP1 phosphorylation appeared to be attenuated by Sestrin2 siRNA. Likewise, Sestrin2 siRNA attenuated leucine-induced S6K phosphorylation in L6 cells. However, Sestrin2 siRNA did not influence leucine-induced S6K phosphorylation in C2C12 cells. ConclusionsIGF-I and leucine cooperatively increased mTORC1 activity in C2C12 cells. IGF-I increased Sestrin2. Sestrin2 siRNA experiments showed that Sestrin2 was involved in the effect of leucine and IGF-I on mTORC1 activity in C2C12 and L6 cells, and suggested that increased Sestrin2 by IGF-I pretreatment might play a role in enhancing the effect of leucine on mTORC1.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines.

Background:Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α-amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines

Background: Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α–amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

Nur77 Increases Glucose Transport in Mouse Skeletal Muscle Cells by Activating p38MAPK Under Lipotoxicity

Diabetes Mellitus (DM) is a common disease in clinic and belongs to a metabolic disease. Related studies have shown that Nur77 regulates sugar metabolism in a variety of tissue cells. Nur77 (orphan nuclear receptor) is a transcription factor and involves in glucose metabolism are different in skeletal muscle cells via p38MAPK signaling pathway. In the study, an IR model was to explore the mechanism of skeletal muscle IR. L6 myocytes were cultured and control, Nur77 knockout and Nur77 overexpression group was set followed by analysis of L6 muscle cell morphology, activity by MTT assay, glucose concentration by glucose oxidase-peroxidase (GOD-POD) p-p38MAPK and skeletal muscle glucose transporter-4 (GLUT4) level by western blot. After 8 to 12 hours of culture, the boundaries between cells were unknown with irregular order and no muscle-shaped structure. The cell morphology was mainly spindle-shaped and triangular. After 2 to 3 days, cell arrangement was regular and morphology was mainly spindle-shaped. The cell activity and glucose concentration in control group at different time points showed no differences which were significantly different in knockout and overexpression group with significantly higher cell activity and glucose concentration for overexpression group than knockout and control group (P &lt; 0.05). In addition, overexpression group also showed significantly increased expression of p-p38MAPK4 and GLUT4. Nur77 can increase glucose transport under lipotoxic state by activating p38MAPK signaling pathway and increasing the protein expression of p-p38MAPK and GLUT4 in muscle cells.

Animal and Cellular Studies Demonstrate Some of the Beneficial Impacts of Herring Milt Hydrolysates on Obesity-Induced Glucose Intolerance and Inflammation.

The search for bioactive compounds from enzymatic hydrolysates has increased in the last few decades. Fish by-products have been shown to be rich in these valuable molecules; for instance, herring milt is a complex matrix composed of lipids, nucleotides, minerals, and proteins. However, limited information is available on the potential health benefits of this by-product. In this context, three industrial products containing herring milt hydrolysate (HMH) were tested in both animal and cellular models to measure their effects on obesity-related metabolic disorders. Male C57Bl/6J mice were fed either a control chow diet or a high-fat high-sucrose (HFHS) diet for 8 weeks and received either the vehicle (water) or one of the three HMH products (HMH1, HMH2, and HMH3) at a dose of 208.8 mg/kg (representing 1 g/day for a human) by daily oral gavage. The impact of HMH treatments on insulin and glucose tolerance, lipid homeostasis, liver gene expression, and the gut microbiota profile was studied. In parallel, the effects of HMH on glucose uptake and inflammation were studied in L6 myocytes and J774 macrophages, respectively. In vivo, daily treatment with HMH2 and HMH3 improved early time point glycemia during the oral glucose tolerance test (OGTT) induced by the HFHS diet, without changes in weight gain and insulin secretion. Interestingly, we also observed that HMH2 consumption partially prevented a lower abundance of Lactobacillus species in the gut microbiota of HFHS diet-fed animals. In addition to this, modulations of gene expression in the liver, such as the upregulation of sucrose nonfermenting AMPK-related kinase (SNARK), were reported for the first time in mice treated with HMH products. While HMH2 and HMH3 inhibited inducible nitric oxide synthase (iNOS) induction in J774 macrophages, glucose uptake was not modified in L6 muscle cells. These results indicate that milt herring hydrolysates reduce some metabolic and inflammatory alterations in cellular and animal models, suggesting a possible novel marine ingredient to help fight against obesity-related immunometabolic disorders.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines.

Background:Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α-amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

Dietary n-3 vs n-6 PUFA Differentially Modulate Macrophage-Myocyte Inflammatory Cross-Talk

Abstract Objectives Skeletal muscle is the primary site for insulin-stimulated glucose uptake. In obesity, increased circulating inflammatory cytokines interfere with skeletal muscle insulin signaling, leading to local and whole-body insulin resistance (IR). Moreover, obese skeletal muscle is characterized by accumulation of infiltrated M1 macrophages and ensuing macrophage-myocyte paracrine interactions (cross-talk) contribute to local inflammation and IR. Such macrophage-myocyte inflammatory cross-talk provides a potential intervention target for anti-inflammatory nutrients, including dietary long-chain n-3 polyunsaturated fatty acids (PUFA). Methods Using a co-culture model designed to mimic the degree of CD11b+ cell accumulation in obese skeletal muscle (40% of immune cells), differentiated L6 myocytes were co-cultured with purified splenic CD11b+ cells from male Sprague Dawley rats (7-wk old) consuming one of three isocaloric diets: i) high-fat (HF; 54% kcal lard, 6% kcal soybean oil), ii) high-fat with n-3 PUFA (HFn-3; 39% kcal lard + 15% kcal menhaden oil + 6% kcal soybean oil) or iii) high-fat with n-6 PUFA (HFn-6; 45% kcal lard + 15% kcal soybean oil) for 2, 8 or 12-wk (n = 8–12/diet). Co-cultures were stimulated for 24 h with lipopolysaccharide (LPS, 10 ng/mL) to mimic in vivo obese endotoxin levels. CD11b+ cells were also cultured alone for 24 h in conditioned media collected from L6 myocytes stimulated with LPS for 24 h (LCM). Results In co-cultures, HFn-6 increased mRNA expression of inflammatory markers compared to HF and HFn-3 at 8- (iNos; P ≤ 0.05) and 12-wk (Tnf-α, Il-6, Il-1β; P ≤ 0.05). Similarly, at 8-wk CD11b+ cells from HFn-6 rats that were treated with LCM, had increased mRNA expression of inflammatory cytokines (Tnf-α, Il-1β) and M1 polarization markers (iNos, Cd86) compared to both HF and HFn-3, and the same effects were seen with Il-6 and Il-1β at 12-wk (P ≤ 0.05). Lastly, HFn-3 reduced mRNA expression of Tnf-α compared to HF at 12-wk (P ≤ 0.05). Conclusions Together, these data suggest that n-6 PUFA support macrophage-myocyte inflammatory cross-talk, in part by promoting M1 macrophage polarization. Further, these data provide mechanistic insight into the benefits of n-3 vs n-6 PUFA inclusion in a high-fat diet in mitigating skeletal muscle inflammation in obesity. Funding Sources Natural Sciences and Engineering Research Council (NSERC) of Canada.

Effects of Retinoic Acid and Insulin on Glycogen Content and Expression Levels of Glycogenic Proteins in Differentiating L6 Myocytes

Abstract Objectives The skeletal muscle is critical for the control of glucose homeostasis partially through glycogenesis. The effects of retinoic acid (RA) on glycogen content and expressions of key glycogenic proteins without or with insulin in L6 rat myocytes deserve to be studied. Methods L6 myocytes at confluence were induced to differentiation using DMEM with 2% horse serum. Cells were incubated in medium with vehicle control or 1 μM RA without or with 10 nM insulin for 2, 4, and 6 days. Glycogen was extracted and analyzed using α-amyloglucosidase and released glucose assay. Proteins in cell lysates from parallel groups were extracted and subjected to analysis using western blotting. The expression levels of glycogen synthase (GS), phospho-glycogen synthase (Ser 641) (p-GS), glycogen synthase kinase 3-β(GSK3β), and phospho-glycogen synthase kinase 3-β (p-GSK3β) were determined using their specific antibodies, and then quantified with ImageJ software. Results The RA + insulin group had higher glycogen content than the control group on days 2 to 6. A synergistic effect of RA and insulin were observed on day 2. GS expression was synergistically induced in the RA + insulin group on each day. The total p-GS normalized to β-actin in the RA + insulin group was higher than that in the control on day 2. However, the ratio of p-GS/GS in the RA + insulin group was lower than that in the control on day 2 and in RA group on day 6, respectively. GSK3β expression levels in insulin and RA + insulin groups were higher than that in the control on day two. The total p-GSK3β normalized to β-actin showed an increase in the insulin group compared to the RA group on day 6. The p-GSK3β/GSK3β ratio in the insulin group was higher than that in RA and RA + insulin groups on day 6. Conclusions Treatments with RA and insulin increase the glycogen content in L6 cells via upregulation of glycogenesis. A synergy of RA and insulin can be seen. The increases of total GS and decreases of p-GS/GS ratio contribute to the elevation of GS activity. RA likely exerts its glycogenic effects through changes of the total GS level and phosphorylation by GSK3β. Funding Sources Internal fund at the Univenrsity of Tennessee.

Blackcurrant Extracts increase Myocyte Proliferation and reduce Apoptosis in L6 myocytes and improve Muscle Strength and Memory in Senescence Accelerated Mice

Musculoskeletal disorders (MSD) impact approximately 50% of adults over the age of 18, and nearly 75% of patients over the age of 65. Sarcopenia is an MSD that is age‐related and is associated with reduced muscle mass and function, as well as and significant morbidity and mortality. Advanced age is associated with significant increase in skeletal muscle cell apoptosis that parallels the loss of both muscle mass and strength. Suppression of myocyte apoptosis can be induced by caloric restriction, exercise training, hormone supplements, drugs and nutritional supplements. Blackcurrants (Ribes nigrum L., Grossulariaceae) are native to Central and Eastern Europe and Northern Asia, and the fruit are used traditionally for the treatment of viral infections. However, the fruit also has potent antioxidant and anti‐inflammatory effects. Blackcurrants contain four major anthocyanins: delphinidin‐3‐O‐glucoside, delphinidin‐3‐O‐rutinoside, cyanidin‐3‐O‐glucoside, cyanidin‐3‐O‐rutinoside. Recent clinical studies reported that blackcurrant extracts improve muscle strength muscle and improve muscle recovery after exercise. In this work, we investigate the effects of blackcurrant extracts and purified anthocyanin compounds on cell proliferation and apoptosis in L6‐rat myoblasts, and in muscle strength and memory in SAMP8 mice. L6 rat myoblasts were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s medium, with 2.5 mM L‐glutamine. Blackcurrant extracts (BCE) were prepared in ethanol and compounds were isolated and purified. Senescence accelerated (SAMP8) mice, a model of senescence acceleration and aging disorders, were from Jackson Laboratories. SAMP mice are susceptible to oxidative stress induced by age‐associated mitochondrial dysfunction and reduced ATP production. SAMP8 mice were maintained under protocol ACC14309 and treated with 100 mg/kg of the BCE for 8 weeks. The results showed that treatment of L6 myocytes with ethanol extracts of R. nigrum fruit increased the proliferation of rat L‐6 myoblasts by 300%, and prevented cellular apoptosis induced by serum and glucose starvation. Of the anthocyanins present in the extract, cyanidin‐3‐glucoside (C3G) was the most active and enhanced L6 growth by 200%, and reduced apoptosis as well. Gene expression analysis showed a concentration‐dependent increase in the anti‐apoptotic Bcl‐2 mRNA expression by ~ 284 fold, as well as enhanced expression of PPARγ by 4 fold. Both BCE and C3G reduced apoptosis by altering the Bax/Bcl‐2 ratio and increased ATP production in favor of cell proliferation. In SAMP8 mice treatment with BCE increased muscle function and improved memory and learning. Overall the data suggest that BCE and C3G have potential for reducing age associated sarcopenia and possibly musculoskeletal disorders such as muscular dystrophy by reducing myocyte apoptosis, increasing ATP production and mitochondrial biogenesis.Support or Funding InformationThis work was supported in part by the Regenstein Foundation (GBM); the Schlumberger Foundation (TOL/GBM); and a Raman Post‐Doctoral Fellowship by the University Grants Commission, Govt. of India to NAR.

Elderberry Extracts increase Rat L6 Myocyte proliferation, reduce Apoptosis and act as HDAC1/SIRT1 Agonists in vitro

Sarcopenia is a geriatric syndrome that is characterized by low muscle mass, strength, and function, that increases morbidity and mortality, and the rates of hospitalization and nursing home admissions in geriatric populations. Poor nutritional intake and alterations in the muscle response to anabolic stimuli from meals in aging populations contribute to the overall pathogenesis of sarcopenia. Research has suggested that there is an increase in skeletal myocyte apoptosis in aging populations. The extent of apoptosis in muscle increases with increasing age, which parallels the loss of both muscle mass and strength. Myocyte apoptosis may be reduced by caloric restriction, exercise training, hormone supplements, drugs and various nutritional supplements. Sambucus nigra L. (Adoxaceae), also known as elderberry is a deciduous tree with blue‐black fruits and cream‐white flowers. Elderberry has been used in traditional systems of medicine to for its antiviral activities, to reduce inflammation and diabetic symptoms. The chemical constituents of elderberry include the flavonoids, such as flavonols, proanthocyanidins and anthocyanins, and simpler phenolic acids. This study investigated the effects of elderberry extracts in rat L6 myoblasts to determine the effects on cell proliferation and apoptosis. L6 cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s medium, with 2.5 mM L‐glutamine (without phenol red). Elderberry extracts were prepared in ethanol and partitions containing anthocyanins were prepared in acetone (SNA) and ethanol (SNE). In L6 cells, ethanol partitions of elderberry increased the proliferation of rat L‐6 myoblasts by 242% and prevented cellular apoptosis induced by serum starvation. The acetone partition increased cell growth by 121% in serum starved L6 cells, the extracts also prevented myoblast apoptosis. Gene expression analysis an increase in the anti‐apoptotic Bcl‐2 mRNA expression and a reduction in Bax expression, thereby reducing the Bax/Bcl‐2 ratio to reduce apoptosis. SNE also increased the expression of PPARγ mRNA by 6.5 fold. Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors belonging to the nuclear receptor superfamily that regulate lipid metabolism, glucose homeostasis, cell proliferation, and differentiation and modulate inflammatory responses. SNE also increased the expression of HDAC1 by 6 fold and SIRT1 by 12 fold. The human Silent Information Regulator Type 1 (SIRT1) is an NAD(+)‐dependent deacetylase that is an intermediary of cellular metabolism in gene silencing and aging. SIRT1 has been extensively investigated and shown to delay senescence and cellular apoptosis. The data suggest that SNE reduces myocyte apoptosis and that the extracts and partitions act as HDAC1/SIRT1 agonists in myocytes.Support or Funding InformationThis work was supported in part by a research grant from the Regenstein Foundation (GBM); a Postdoctoral fellowship award from the Schlumberger Foundation (TOL/GBM); and a Raman Post‐Doctoral Fellowship by the University Grants Commission, Govt. of India to NAR.

Assessment of the antidiabetic potentiality of glyburide loaded glyceryl monostearate solid lipid nanoparticles

The high rate homogenisation techniques was used for preparing glyburide loaded glyceryl monostearate solid lipid nanoparticles (Glb-SLNs) to improve the drug's solubility and oral bioavailability. It was mediated through pseudo-ternary phase diagrams to optimize the surfactant/co-surfactant ratio. The optimized Glb-SLNs were characterized by FT-IR spectroscopy, DSC, powder X-ray diffraction study and SEM analysis. In rat skeletal muscle cells (L6 myocytes) and in streptozotocin and high-fat diet-induced diabetic rats, anti-diabetic activity was evaluated. Flow cytometry analysis has accessed the effects of glucose uptake performed by the 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2- deoxyglucose (2-NBDG) in L6 myotubes. In normal rats and diabetic rats, parameters such as the blood glucose level, serum insulin, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), lipid peroxidase, catalase, glutathione, and HbA1c levels were assessed. Glb-SLNs at a dose of 50 μM showed a relative increase in relative fluorescence intensity of 92.5% to represent the glucose uptake potentiality. In vivo results revealed that the treatment with SLNs of glyburide (Glb-SLNs) normalized blood-glucose levels and serum biochemical parameters as compared with those of STZ controls. The protective effect was further confirmed by histopathological examination of the liver. These animals showed a comparable histopathological regeneration on Glb-SLNs treatment. These positive results demonstrated the success of the proposed SLNs approach in enhancing glyburide oral bioavailability and confirmed its potential as an effective system for diabetes treatment.

Flavonoids extracted from mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes.

Mulberry (Morus alba L.) leaves have been widely applied to controlling blood glucose as a efficacious traditional Chinese medicine or salutary medical supplement. The extracts of mulberry leaf suppress inflammatory mediators and oxidative stress, protect the pancreatic β-cells and modulate glucose metabolism in diabetic rats. Our previous studies and others have shown that mulberry leaf extract has excellent therapeutic effects on type 2 diabetes mellitus (T2DM), however, the underlying mechanism remains to be studied. Skeletal muscle insulin resistance (IR) plays an important role in the pathogenesis of T2DM. The aim of this study was to investigate the effects and mechanisms of Mulberry leaf flavonoids (MLF) in L6 skeletal muscle cells and db/db mice. L6 skeletal muscle cells were cultured and treated with/without MLF for in vitro studies. For in vivo studies, the db/db mice with/without MLF therapy were used. Coomassie brilliant blue staining and α-SMA immunofluorescence staining were used to identify the differentiated L6 cells. Glucose level and ATP level of L6 myotubes were performed by optical density detection and cell viability was performed by MTT method. Mitochondrial membrane potential of L6 myotubes was detected by JC-1 fluorescent staining. ROS level of L6 myotubes was detected by DCFH-DA fluorescent staining. The body weight, food intake, and blood glucose of the mice were measured in different treatment days. Oral glucose tolerance test (OGTT), starch glucose tolerance test (STT) and insulin tolerance test (ITT) were performed in mice. Glycated hemoglobin, glycated serum protein, insulin, liver and muscle glycogen, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) of the mice were detected by corresponding kit. The pathologic change of pancreas and skeletal muscle of mice were performed by H & E staining. Immunohistochemistry staining was used to detect the GLUT4 and p-AMPK expressions in skeletal muscle in mice. GLUT4, CPT-1, NRF1, COXIV, PGC-1α, and p-AMPK expression levels in L6 cells and mice were detected by western bolt assay. MLF and metformin significantly ameliorated muscle glucose uptake and mitochondrial function in L6 muscle cells. MLF also increased the phosphorylation of AMPK and the expression of PGC-1α, and up-regulated the protein levels of m-GLUT4 and T-GLUT4. These effects were reversed by the AMPK inhibitor compound C. In db/db mice, MLF improve diabetes symptoms and insulin resistance. Moreover, MLF elevated the levels of p-AMPK and PGC-1α, raised m-GLUT4 and T-GLUT4 protein expression, and ameliorated mitochondrial function in skeletal muscle of db/db mice. MLF significantly improved skeletal muscle insulin resistance and mitochondrial function in db/db mice and L6 myocytes through AMPK-PGC-1α signaling pathway, and our findings support the therapeutic effects of MLF on type 2 diabetes.

Abstract 361: Deacetylation of Lc3 Drives Autophagy and Proteome Remodeling in Skeletal Muscle During Oncometabolic Stress

Metabolic rewiring is a hallmark of cancer and muscle cells. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. Our recent in vivo work showed that increased D2-HG supply by IDH-mutant cells causes heart and skeletal muscle atrophy, and decreases cellular ATP and NADH. Although heart failure and cachexia in cancer are commonly associated with chemotherapy, cancer survivors have a 5-fold increased risk of heart failure independent of any cytostatic treatment. The connection between metabolic and proteomic remodeling in this context remain poorly understood. We hypothesize that D2-HG-mediated alpha-ketoglutarate dehydrogenase (AKGDH) inhibition in myocytes results in metabolomic perturbations, increases autophagy and proteomic remodeling. Here, we report that LC3, a key regulator of autophagy, is activated in the nucleus of myocytes in presence of D2-HG through deacetylation by the nuclear deacetylase Sirt1. Activation of Sirt1 is driven by increased NAD + levels through D2-HG-mediated AKGDH inhibition. We used LC3 mutants with arginine and glutamine replacements at lysine residues to show that deacetylation of LC3 at K49 and K51 by Sirt1 shifts LC3 distribution from the nucleus into the cytosol, where it is able to undergo lipidation at pre-autophagic membranes. Live cell imaging with GFP-tagged LC3 in L6 myocytes indicated that the cycle of acetylation-deacetylation allows LC3 to redistribute from the nucleus to the cytosol within less than 24 h. Co-immunoprecipitation of LC3 followed by proteomics analysis revealed that LC3 binds to dynein in presence of D2-HG. Furthermore, D2-HG promoted skeletal muscle atrophy and reduced grip strength in wild-type C57BL/J6 mice in vivo. Using LC-MS/MS-based proteomics and metabolomics combined with RNA-sequencing, we assessed the effect of D2-HG on a systems level in skeletal muscle. Pathway-enrichment analysis revealed that D2-HG induces upregulation of key metabolic enzymes involved in glycolysis and the pentose phosphate pathway. In short, autophagy activation supports proteome remodeling in muscle cells during IDH-mutant leukemia.