The Lipophilic Extract from Ginkgo biloba L. Leaves Promotes Glucose Uptake and Alleviates Palmitate-Induced Insulin Resistance in C2C12 Myotubes.

Ginkgo biloba L. (ginkgo) is a widely used medicinal plant around the world. Its leaves, which have been used as a traditional Chinese medicine, are rich in various bioactive components. However, most of the research and applications of ginkgo leaves have focused on terpene trilactones and flavonol glycosides, thereby overlooking the other active components. In this study, a lipophilic extract (GL) was isolated from ginkgo leaves. This extract is abundant in lipids and lipid-like molecules. Then, its effect and potential mechanism on glucose uptake and insulin resistance in C2C12 myotubes were investigated. The results showed that GL significantly enhanced the translocation of GLUT4 to the plasma membrane, which subsequently promoted glucose uptake. Meanwhile, it increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream targets. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor compound C reversed these effects. Additionally, GL ameliorated palmitate-induced insulin resistance by enhancing insulin-stimulated glucose uptake, increasing the phosphorylation of protein kinase B (PKB/AKT), and restoring the translocation of GLUT4 from the cytoplasm to the membrane. However, pretreatment with compound C abolished these beneficial effects of GL. In conclusion, GL enhances basal glucose uptake in C2C12 myotubes and improves insulin sensitivity in palmitate-induced insulin resistant myotubes through the AMPK pathway.

Gracilaria chorda subcritical-water extracts as ameliorant of insulin resistance induced by high-glucose in zebrafish and dexamethasone in L6 myotubes.

Background and aim: Insulin resistance (IR) is a pathological condition in which cells fail to respond normally to insulin. Loss of insulin sensitivity disrupts glucose homeostasis and elevates the risk of developing the metabolic syndrome that includes Type 2 diabetes. This study assesses the effect on subcritical-water extract of Gracilaria chorda (GC) at 210 °C (GCSW210) in IR induction models of high glucose (HG)-induced zebrafish larvae and dexamethasone (DEX)-induced L6 myotubes. Experimental procedure: The dose of HG and DEX for IR induction in zebrafish larvae and L6 myotubes was 130 mM or 0.5 µM. The capacity of glucose uptake was quantified by fluorescence staining or intensity. In addition, the activation of protein and mRNA expressions for insulin signaling (insulin-dependent or independent pathways) was measured. Results and conclusion: Exposure of zebrafish larvae to HG significantly reduced the intracellular glucose uptake with dose-dependnet manner compared to control. However, the group treated with GCSW210 significantly averted HG levels like the insulin-treated group, and significantly up- or down-regulated the mRNA expressions related to insulin production (insα) and insulin signaling pathways. Moreover, the treatment with GCSW210 effectively regulated the protein expression of PI3K/AKT, AMPK, and GLUT4 involved in the action of insulin in IR models of L6 myotubes compared to DEX-treated control. Our data indicate that GCSW210 stimulates activation of PI3K/AKT and AMPK pathways to attenuate the development of IR induced by HG in zebrafish and DEX in L6 myotubes. In conclusion, GCSW210 is a potential agent for alleviating various diseases associated with the insulin resistance.

7ß-Hydroxycholesterol and 7-ketocholesterol: New oxidative stress biomarkers of sarcopenia inducing cytotoxic effects on myoblasts and myotubes.

Aging is a complex biological process which can be associated with skeletal muscle degradation leading to sarcopenia. The aim of this study consisted i) to determine the oxidative and inflammatory status of sarcopenic patients and ii) to clarify the impact of oxidative stress on myoblasts and myotubes. To this end, various biomarkers of inflammation (C-reactive protein (CRP), TNF-α, IL-6, IL-8, leukotriene B4 (LTB4)) and oxidative stress (malondialdehyde, conjugated dienes, carbonylated proteins and antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase) as well as oxidized derivatives of cholesterol formed by cholesterol autoxidation (7-ketocholesterol, 7ß-hydroxycholesterol), were analyzed. Apelin, a myokine which contributes to muscle strength, was also quantified. To this end, a case-control study was conducted to evaluate the RedOx and inflammatory status in 45 elderly subjects (23 non-sarcopenic; 22 sarcopenic) from 65 years old and higher. SARCopenia-Formular (SARC-F) and Timed Up and Go (TUG) tests were used to distinguish between sarcopenic and non-sarcopenic subjects. By using red blood cells, plasma and/or serum, we observed in sarcopenic patients an increased activity of major antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) associated with lipid peroxidation and protein carbonylation (increased level of malondialdehyde, conjugated dienes and carbonylated proteins). Higher levels of 7-ketocholesterol and 7ß-hydroxycholesterol were also observed in the plasma of sarcopenic patients. Significant differences were only observed with 7ß-hydroxycholesterol. In sarcopenic patients comparatively to non-sarcopenic subjects, significant increase of CRP, LTB4 and apelin were observed whereas similar levels of TNF-α, IL-6 and IL-8 were found. The increased plasma level of 7-ketocholesterol and 7ß-hydroxycholesterol in sarcopenic patients led us to study the cytotoxic effect of these oxysterols on undifferentiated (myoblasts) and differentiated (myotubes) murine C2C12 cells. With the fluorescein diacetate and sulforhodamine 101 assays, an induction of cell death was observed both on undifferentiated and differentiated cells: the cytotoxic effects were less pronounced with 7-ketocholesterol. In addition, IL-6 secretion was never detected whatever the culture conditions, TNF-α secretion was significantly increased on undifferentiated and differentiated C2C12 cells treated with 7-ketocholesterol- and 7ß-hydroxycholesterol, and IL-8 secretion was increased on differentiated cells. 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cell death was strongly attenuated by α-tocopherol and Pistacia lentiscus L. seed oil both on myoblasts and/or myotubes. TNF-α and/or IL-8 secretions were reduced by α-tocopherol and Pistacia lentiscus L. seed oil. Our data support the hypothesis that the enhancement of oxidative stress observed in sarcopenic patients could contribute, especially via 7ß-hydroxycholesterol, to skeletal muscle atrophy and inflammation via cytotoxic effects on myoblasts and myotubes. These data bring new elements to understand the pathophysiology of sarcopenia and open new perspectives for the treatment of this frequent age-related disease.

NMR-Based Metabolic Profiling of the Effects of α-Ketoglutarate Supplementation on Energy-Deficient C2C12 Myotubes.

Skeletal muscle is closely linked to energy metabolism, but it is inevitably deprived of energy. Cellular differentiation is an essential and energy-demanding process in skeletal muscle development. Much attention has been paid to identifying beneficial factors that promote skeletal muscle satellite cell differentiation and further understanding the underlying regulatory mechanisms. As a critical metabolic substrate or regulator, α-ketoglutarate (AKG) has been recognized as a potential nutritional supplement or therapeutic target for skeletal muscle. We have previously found beneficial effects of AKG supplementation on the proliferation of C2C12 myoblasts cultured under both normal and energy-deficient conditions and have further elucidated the underlying metabolic mechanisms. However, it remains unclear what role AKG plays in myotube formation in different energy states. In the present study, we investigated the effects of AKG supplementation on the differentiation of C2C12 myoblasts cultured in normal medium (Nor myotubes) and low glucose medium (Low myotubes) and performed NMR-based metabonomic profiling to address AKG-induced metabolic changes in both Nor and Low myotubes. Significantly, AKG supplementation promoted myotube formation and induced metabolic remodeling in myotubes under normal medium and low glucose medium, including improved energy metabolism and enhanced antioxidant capacity. Specifically, AKG mainly altered amino acid metabolism and antioxidant metabolism and upregulated glycine levels and antioxidase expression. Our results are typical for the mechanistic understanding of the effects of AKG supplementation on myotube formation in the two energy states. This study may be beneficial for further exploring the applications of AKG supplementation in sports, exercise, and therapy.

Chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) leaf extracts regulate mitochondrial bioenergetics and fatty acid oxidation in C2C12 myotubes and primary hepatocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) is an important component of the regular diet and traditional medicine of indigenous communities in Mexico. Customarily, Chaya is consumed as a beverage made of macerated leaf, cooked, or prepared in teas or infusions to empirically treat obesity, diabetes, gastrointestinal disorders, and kidney stones. The beneficial effects of Chaya can be attributed to the presence of protein, dietary fiber, vitamins, and especially polyphenols, which regulate mitochondrial function. Therefore, polyphenols present in Chaya extracts could be used to develop novel strategies to prevent and treat metabolic alterations related to mitochondrial dysfunction in the muscle and liver of subjects with obesity, type 2 diabetes, and cardiovascular diseases. However, limited information is available concerning the effect of Chaya extracts on mitochondrial activity in those tissues. AIM OF THE STUDY: The aim of this study was to evaluate the antioxidant capacity of an aqueous extract (AE) or mixed (methanol/acetone/water) extract (ME) of Chaya leaf and their effect on C2C12 myotubes and primary hepatocyte mitochondrial bioenergetics and fatty acid oxidation (FAO). MATERIALS AND METHODS: Total polyphenol content and antioxidant activity were determined using the Folin-Ciocalteu method and the oxygen radical absorbance capacity assay, respectively. The effect of AE and ME from Chaya leaf on mitochondrial activity and FAO of C2C12 myotubes and primary hepatocytes was evaluated using an extracellular flux analyzer. RESULTS: The AE and ME from Chaya leaf exhibited antioxidant activity and a polyphenol content similar to nopal, another plant used in Mexican traditional medicine. AE significantly (p < 0.05) decreased the maximal respiration and spare respiratory capacity (SRC) of C2C12 cells, whereas ME had little effect on C2C12 mitochondrial function. Conversely, ME significantly (p < 0.05) decreased SRC in primary hepatocytes, whereas AE increased maximal respiration and SRC at low doses (5 and 10 µM). Moreover, low doses of Chaya AE significantly (p < 0.05) increased AMPK phosphorylation, acyl-coenzyme A oxidase protein abundance, and palmitate oxidation in primary hepatocytes. CONCLUSION: The AE of Chaya leaf increases mitochondrial function and FAO of primary hepatocytes, indicating its potential to treat hepatic mitochondrial dysfunction underlying metabolic diseases.

LCMS Determination and Cytotoxicity of Abrus precatorius on L6 and SK-N-MC Cell Lines.

OBJECTIVE: The present study aimed to investigate the cytotoxic effect of various extracts derived from Abrus precatorius Linn. leaves on rat L6 and human SK-N-MC neuroblastoma cell lines and determine the secondary metabolites responsible for the cytotoxicity of Abrus precatorius. METHODS: Successive solvent extraction of A. precatorius leaves was carried out using the Soxhlet apparatus with solvents such as petroleum ether, chloroform, ethyl acetate, and ethanol. HPTLC fingerprinting and LC-MS studies were performed to assess the presence of secondary metabolites, such as flavonoids and phenols, in the ethyl acetate extract. Furthermore, the cytotoxic effect of extracts was tested on rat skeletal muscle cell line L6 and human neuroblastoma cell line SK-N-MC using MTT assay. RESULTS: The total phenolic content of ethyl acetate and ethanol extracts of A. precatorius were 72.67 and 60.73 mg, respectively, of GAE/g dry weight of the extract. The total flavonoid content of ethyl acetate and ethanol extract of A. precatorius were 107.33 and 40.66 mg of Quercetin equivalents/g dry weight of the extract. LCMS analysis demonstrated that the flavonoids in specific Naringenin, Diosmetin, Glycitin, and Genistein might play a prominent role in the cytotoxicity of A. precatorius. The cytotoxicity study revealed that the extracts of A. precatorius were non-toxic to rat L6 myotubes, and the IC50 values of the various extracts, such as APPE, APCH, APEA, and APET, were >100 µg/ml. The extracts exhibited cytotoxic activity against human neuroblastoma SK-N-MC cells, and the IC50 values of APPE, APCH, APEA, APET, and the standard drug "Cisplatin" were >100, >100, 64.88, >100, and 3.72 µg/ml, respectively. CONCLUSION: It was concluded from the study that the extracts of Abrus precatorius were cytotoxic to neuroblastoma cell lines but non-toxic to normal cell lines. HPTLC and LC-MS studies confirmed that flavonoids in the ethyl acetate extract could be responsible for the biological activity.

In vitro chemotherapy-associated muscle toxicity is attenuated with nutritional support, while treatment efficacy is retained.

PURPOSE: Muscle-wasting and treatment-related toxicities negatively impact prognosis of colorectal cancer (CRC) patients. Specific nutritional composition might support skeletal muscle and enhance treatment support. In this in vitro study we assess the effect of nutrients EPA, DHA, L-leucine and vitamin D3, as single nutrients or in combination on chemotherapy-treated C2C12-myotubes, and specific CRC-tumor cells. MATERIALS AND METHODS: Using C2C12-myotubes, the effects of chemotherapy (oxaliplatin, 5-fluorouracil, oxaliplatin+5-fluorouracil and irinotecan) on protein synthesis, cell-viability, caspase-3/7-activity and LDH-activity were assessed. Addition of EPA, DHA, L-leucine and vitamin D3 and their combination (SNCi) were studied in presence of above chemotherapies. Tumor cell-viability was assessed in oxaliplatin-treated C26 and MC38 CRC cells, and in murine and patient-derived CRC-organoids. RESULTS: While chemotherapy treatment of C2C12-myotubes decreased protein synthesis, cell-viability and increased caspase-3/7 and LDH-activity, SNCi showed improved protein synthesis and cell viability and lowered LDH activity. The nutrient combination SNCi showed a better overall performance compared to the single nutrients. Treatment response of tumor models was not significantly affected by addition of nutrients. CONCLUSIONS: This in vitro study shows protective effect with specific nutrition composition of C2C12-myotubes against chemotherapy toxicity, which is superior to the single nutrients, while treatment response of tumor cells remained.

Laurel Attenuates Dexamethasone-Induced Skeletal Muscle Atrophy In Vitro and in a Rat Model.

Prevention of muscle atrophy contributes to improved quality of life and life expectancy. In this study, we investigated the effects of laurel, selected from 34 spices and herbs, on dexamethasone (DEX)-induced skeletal muscle atrophy and deciphered the underlying mechanisms. Co-treatment of C2C12 myotubes with laurel for 12 h inhibited the DEX-induced expression of intracellular ubiquitin ligases-muscle atrophy F-box (atrogin-1/MAFbx) and muscle RING finger 1 (MuRF1)-and reduction in myotube diameter. Male Wistar rats were supplemented with 2% laurel for 17 days, with DEX-induced skeletal muscle atrophy occurring in the last 3 days. Laurel supplementation inhibited the mRNA expression of MuRF1, regulated DNA damage and development 1 (Redd1), and forkhead box class O 1 (Foxo1) in the muscles of rats. Mechanistically, we evaluated the effects of laurel on the cellular proteolysis machinery-namely, the ubiquitin/proteasome system and autophagy-and the mTOR signaling pathway, which regulates protein synthesis. These data indicated that the amelioration of DEX-induced skeletal muscle atrophy induced by laurel, is mainly mediated by the transcriptional inhibition of downstream factors of the ubiquitin-proteasome system. Thus, laurel may be a potential food ingredient that prevents muscle atrophy.

Effects of American wild ginseng and Korean cultivated wild ginseng pharmacopuncture extracts on the regulation of C2C12 myoblasts differentiation through AMPK and PI3K/Akt/mTOR signaling pathway.

Targeting impaired myogenesis and mitochondrial biogenesis offers a potential alternative strategy for balancing energy to fight muscle disorders such as sarcopenia. In traditional Korean medicine, it is believed that the herb wild ginseng can help restore energy to the elderly. The present study investigated whether American wild ginseng pharmacopuncture (AWGP) and Korean cultivated wild ginseng pharmacopuncture (KCWGP) regulate energy metabolism in skeletal muscle cells. C2C12 mouse myoblasts were differentiated into myotubes using horse serum for 5 days. An MTT colorimetric assay verified cell viability. AWGP, KCWGP (0.5, 1, or 2 mg/ml), or metformin (2.5 mM) for reference were used to treat the C2C12 myotubes. The expressions of differentiation and mitochondrial biogenetic factors were measured by western blotting in C2C12 myotubes. Treatment of C2C12 cells stimulated with AWGP and KCWGP at a concentration of 10 mg/ml did not affect cell viability. AWGP and KCWGP treatments resulted in significant increases in the myogenesis proteins, myosin heavy chain, myostatin, myoblast determination protein 1 and myogenin, as well as increases to the biogenic regulatory factors, peroxisome proliferator­activated receptor­Î³ coactivator­1­α, nuclear respiratory factor 1, mitochondrial transcription factor A and Sirtuin 1, in the myotubes through AMPK and PI3K/AKT/mTOR signaling pathway activation. These results suggest that AWGP and KCWGP may be beneficial to muscle function by improving muscle differentiation and energy metabolism.

Naringin induces skeletal muscle fiber type transformation via AMPK/PGC-1α signaling pathway in mice and C2C12 myotubes.

A large number of studies have shown that polyphenols can regulate skeletal muscle fiber type transformation through AMPK signal. However, the effects and mechanism of naringin (a natural polyphenol) on muscle fiber type transformation still remains unclear. Thus, we hypothesized that naringin would induce the transformation of skeletal muscle fibers from type II to type I by AMPK signaling. C2C12 myotubes and BALB/c mice models were used to test this hypothesis. We found that naringin significantly increased the protein expression of slow myosin heavy chain (MyHC), myoglobin and troponin I type I slow skeletal (Troponin I-SS) and the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), and significantly decreased fast MyHC protein expression and lactate dehydrogenase (LDH) activity, accompanied by the activation of AMPK and the activity of peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) in mice and C2C12 myotubes. Further inhibition of AMPK activity by compound C showed that the above effects were significantly inhibited in C2C12 myotubes. In conclusion, naringin promotes the transformation of skeletal muscle fibers from type II to type I through AMPK/PGC-1α signaling pathway, which not only enriches the nutritional and physiological functions of naringin, but also provides a theoretical basis for the regulation of muscle fiber type transformation by nutritional approaches.

14-Deoxy-11,12-Didehydroandrographolide Ameliorates Glucose Intolerance Enhancing the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 Signaling Pathway and Inducing GLUT4 Expression in Myotubes and Skeletal Muscle of Obese Mice.

14-Deoxy-11,12-didehydroandrographolide (deAND), a bioactive component of Andrographis paniculata, has antidiabetic activity. AMP-activated protein kinase (AMPK) regulates glucose transport and ameliorates insulin resistance. The aim of the present study was to investigate whether activation of AMPK is involved in the mechanism by which deAND ameliorates insulin resistance in muscles. deAND amounts up to 40 [Formula: see text]M dose-dependently activated phosphorylation of AMPK[Formula: see text] and TBC1D1 in C2C12 myotubes. In addition, deAND significantly activated phosphorylation of LKB1 at 6 h after treatment, and this activation was maintained up to 48 h. deAND increased glucose uptake at 18 h after treatment, and this increase was time dependent up to 72 h. Compound C, an inhibitor of AMPK, suppressed deAND-induced phosphorylation of AMPK[Formula: see text] and TBC1D1 and reversed the effect on glucose uptake. In addition, the expression of GLUT4 mRNA and protein in C2C12 myotubes was up-regulated by deAND in a time-dependent manner. Promotion of GLUT4 gene transcription was verified by a pGL3-GLUT4 (837 bp) reporter assay. deAND also increased the nuclear translocation of MEF-2A and PPAR[Formula: see text]. After 16 weeks of feeding, the high-fat diet (HFD) inhibited phosphorylation of AMPK[Formula: see text] and TBC1D1 in skeletal muscle of obese C57BL/6JNarl mice, and deactivation of AMPK[Formula: see text] and TBC1D1 by the HFD was abolished by deAND supplementation. Supplementation with deAND significantly promoted membrane translocation of GLUT4 compared with the HFD group. Supplementation also significantly increased GLUT4 mRNA and protein expression in skeletal muscle compared with the HFD group. The hypoglycemic effects of deAND are likely associated with activation of the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 signaling pathway and stimulation of MEF-2A- and PPAR[Formula: see text]-dependent GLUT4 gene expression, which account for the glucose uptake into skeletal muscle and lower blood glucose levels.

Coenzyme Q nanodisks counteract the effect of statins on C2C12 myotubes.

Depletion of coenzyme Q (CoQ) is associated with disease, ranging from myopathy to heart failure. To induce a CoQ deficit, C2C12 myotubes were incubated with high dose simvastatin. This resulted in a concentration-dependent inhibition of cell viability. Simvastatin-induced effects were prevented by co-incubation with mevalonic acid. When myotubes were incubated with 60 µM simvastatin, mitochondrial CoQ content decreased while co-incubation with CoQ nanodisks (ND) increased mitochondrial CoQ levels and improved cell viability. Incubation of myotubes with simvastatin also led to a reduction in oxygen consumption rate (OCR). When myotubes were co-incubated with simvastatin and CoQ ND, the decline in OCR was ameliorated. The data indicate that CoQ ND represent a water soluble vehicle capable of delivering CoQ to cultured myotubes. Thus, these biocompatible nanoparticles have the potential to bypass poor CoQ oral bioavailability as a treatment option for individuals with severe CoQ deficiency syndromes and/or aging-related CoQ depletion.

Prenylflavonoids from fruit of Macaranga tanarius promote glucose uptake via AMPK activation in L6 myotubes.

Skeletal muscle is a major tissue of glucose consumption and plays an important role in glucose homeostasis. Prenylflavonoids, a component of Macaranga tanarius fruits, have been reported to have antioxidant, antibacterial, and anticancer effects. However, the effects of these compounds on skeletal muscle glucose metabolism are unclear. Here, we isolated five prenylflavonoids from M. tanarius fruits, and investigated the mechanism of action of these compounds on skeletal muscle cells using L6 myotubes. We found that isonymphaeol B and 3'-geranyl naringenin increased glucose uptake in a dose-dependent manner. Furthermore, both isonymphaeol B and 3'-geranyl naringenin increased AMPK phosphorylation but did not affect PI3K-Akt phosphorylation. Isonymphaeol B and 3'-geranyl naringenin also increased Glut1 mRNA expression and plasma membrane GLUT1 protein levels. These results suggest that isonymphaeol B and 3'-geranyl naringenin have beneficial effects on glucose metabolism through AMPK and GLUT1 pathway. Isonymphaeol B and 3'-geranyl naringenin may be potential lead candidates for antidiabetic drug development.

Effects of Physiological Doses of Resveratrol and Quercetin on Glucose Metabolism in Primary Myotubes.

Phenolic compounds have emerged in recent years as an option to face insulin resistance and diabetes. The central aim of this study was: (1) to demonstrate that physiological doses of resveratrol (RSV) or quercetin (Q) can influence glucose metabolism in human myotubes, (2) to establish whether AMP-activated protein kinase (AMPK) and protein kinase B -PKB- (Akt) pathways are involved in this effect. In addition, the effects of these polyphenols on mitochondrial biogenesis and fatty acid oxidation were analysed. Myotubes from healthy donors were cultured for 24 h with either 0.1 µM of RSV or with 10 µM of Q. Glucose metabolism, such as glycogen synthesis, glucose oxidation, and lactate production, were measured with D[U-14C]glucose. ß-oxidation using [1-14C]palmitate as well as the expression of key metabolic genes and proteins by Real Time PCR and Western blot were also assessed. Although RSV and Q increased pgc1α expression, they did not significantly change either glucose oxidation or ß-oxidation. Q increased AMPK, insulin receptor substrate 1 (IRS-1), and AS160 phosphorylation in basal conditions and glycogen synthase kinase 3 (GSK3ß) in insulin-stimulated conditions. RSV tended to increase the phosphorylation rates of AMPK and GSK3ß. Both of the polyphenols increased insulin-stimulated glycogen synthesis and reduced lactate production in human myotubes. Thus, physiological doses of RSV or Q may exhibit anti-diabetic actions in human myotubes.

Flavanols and triterpenoids from Myrianthus arboreus ameliorate hyperglycaemia in streptozotocin-induced diabetic rats possibly via glucose uptake enhancement and α-amylase inhibition.

Myrianthus arboreus is use traditionally as an antidiabetic agent in Ghana. We reported the in vivo antidiabetic activity of its 70 % ethanol stem bark extract (MAB) which we found to be strongly concentrated in its EtOAc fraction using glucose uptake and enzyme inhibitory assays. The present study sought to investigate the in vivo hypoglycaemic and anti-hyperlipidaemic activity of this ethyl acetate fraction of MAB (MAB-EtOAc, 50 and 100 mg/kg) in streptozotocin (STZ)-induced diabetic rats for 21 days, isolate and evaluate the bioactive constituents responsible for the antidiabetic activity. In silico pharmacokinetic and toxicity properties of the most active compound was also determined. MAB-EtOAc significantly (p < 0.001) reduced the blood glucose levels while normalizing considerably the altered serum lipid parameters of the diabetic rats which was comparable to glibenclamide (5 mg/kg). Chemical investigation of MAB-EtOAc led to the isolation of seven known compounds including three flavanols which are reported for the first time in the plant: epicatechin (1), epigallocatechin (2), dulcisflavan (3), euscaphic acid (4), tormentic acid (5), sitosterol-3-O-ß-d-glucopyranoside (6) and arjunolic acid (7). The compounds markedly inhibited the action of α-amylase and, except for 4 and 6, which stimulated considerably glucose uptake in C2C12 cells. Compounds 2, 3, 5, 6 and 7 which were further evaluated in STZ-induced diabetic rats demonstrated hypoglycaemic and anti-hyperlipidaemic activities which, however, were not comparable with MAB-EtOAc. Compound 3, the most active compound was predicted to be non-toxic, non-mutagenic, has reasonable oral bioavailability and a decent substrate for further drug development. The findings of this study show that the isolated compounds may contribute to the antidiabetic activity of M. arboreus and could serve as marker compounds for the quality control of herbal medicines that would be made from the plant.

Pimarane Diterpenoids from the Seeds of Caesalpinia minax as PTP1B Inhibitors and Insulin Sensitizers.

Protein-tyrosine phosphatase 1B (PTP1B) has been considered as a promising target for treating insulin resistance. In searching for naturally occurring PTB1B antagonists, two new pimarane diterpenoids, named 2α-hydroxy-7-oxo-pimara-8(9),15-diene (1) and 19-hydroxy-2α-acetoxy-7-oxo-pimara-8(9),15-diene (2), were isolated from the seeds of Caesalpinia minax. Their structures were determined by extensive analysis of NMR and HR-ESIMS data, and their absolute configurations were determined by electronic circular dichroism (ECD) spectra. Compound 1 was disclosed as a competitive inhibitor of PTP1B with an IC50 (the half-maximal inhibitory concentration) value of 19.44 ± 2.39 µM and a Ki (inhibition constant) value of 13.69 ± 2.72 µM. Moreover, compound 1 dose-dependently promoted insulin-stimulated glucose uptake in C2C12 myotubes through activating insulin signaling pathway. Compound 1 might be further developed as an insulin sensitizer.

Thirty years of translational research in Mobility Medicine: Collection of abstracts of the 2020 Padua Muscle Days.

More than half a century of skeletal muscle research is continuing at Padua University (Italy) under the auspices of the Interdepartmental Research Centre of Myology (CIR-Myo), the European Journal of Translational Myology (EJTM) and recently also with the support of the A&CM-C Foundation for Translational Myology, Padova, Italy. The Volume 30(1), 2020 of the EJTM opens with the collection of abstracts for the conference "2020 Padua Muscle Days: Mobility Medicine 30 years of Translational Research". This is an international conference that will be held between March 18-21, 2020 in Euganei Hills and Padova in Italy. The abstracts are excellent examples of translational research and of the multidimensional approaches that are needed to classify and manage (in both the acute and chronic phases) diseases of Mobility that span from neurologic, metabolic and traumatic syndromes to the biological process of aging. One of the typical aim of Physical Medicine and Rehabilitation is indeed to reduce pain and increase mobility enough to enable impaired persons to walk freely, garden, and drive again. The excellent contents of this Collection of Abstracts reflect the high scientific caliber of researchers and clinicians who are eager to present their results at the PaduaMuscleDays. A series of EJTM Communications will also add to this preliminary evidence.

Alisol A-24-acetate promotes glucose uptake via activation of AMPK in C2C12 myotubes.

BACKGROUND: Alisol A-24-acetate (AA-24-a) is one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., which possesses multiple biological activities, including a hypoglycemic effect. Whether AA-24-a is a hypoglycemic-active compound of A. orientale (Sam.) Juz. is unclear. The present study aimed to clarify the effect and potential mechanism of action of AA-24-a on glucose uptake in C2C12 myotubes. METHOD: Effects of AA-24-a on glucose uptake and GLUT4 translocation to the plasma membrane were evaluated. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. To determine the underlying mechanism, the phosphorylation levels of proteins involved in the insulin and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined using western blotting. Furthermore, specific inhibitors of key enzymes in AMPK signaling pathway were used to examine the role of these kinases in the AA-24-a-induced glucose uptake and GLUT4 translocation. RESULTS: We found that AA-24-a significantly promoted glucose uptake and GLUT4 translocation in C2C12 myotubes. AA-24-a increased the phosphorylation of AMPK, but had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/AKT). In addition, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the AKT substrate of 160 kDa (AS160), two proteins that act downstream of AMPK, was upregulated. Compound C, an AMPK inhibitor, blocked AA-24-a-induced AMPK pathway activation and reversed AA-24-a-induced glucose uptake and GLUT4 translocation to the plasma membrane, indicating that AA-24-a promotes glucose metabolism via the AMPK pathway in vitro. STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, also attenuated AA-24-a-induced glucose uptake and GLUT4 translocation. Moreover, STO-609 weakened AA-24-a-induced phosphorylation of AMPK, p38 MAPK and AS160. CONCLUSIONS: These results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the CaMKKß-AMPK-p38 MAPK/AS160 pathway.

Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes.

Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes typically display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease commonly manifests at later stages of development. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by the expression profile of myosin heavy chain isoforms, as well as the upregulation of genes related with muscle contractile function. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle constructs, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.