Free essential amino acid feeding improves endurance during resistance training via DRP1-dependent mitochondrial remodelling.

BACKGROUND: Loss of muscle strength and endurance with aging or in various conditions negatively affects quality of life. Resistance exercise training (RET) is the most powerful means to improve muscle mass and strength, but it does not generally lead to improvements in endurance capacity. Free essential amino acids (EAAs) act as precursors and stimuli for synthesis of both mitochondrial and myofibrillar proteins that could potentially confer endurance and strength gains. Thus, we hypothesized that daily consumption of a dietary supplement of nine free EAAs with RET improves endurance in addition to the strength gains by RET. METHODS: Male C57BL6J mice (9 weeks old) were assigned to control (CON), EAA, RET (ladder climbing, 3 times a week), or combined treatment of EAA and RET (EAA + RET) groups. Physical functions focusing on strength or endurance were assessed before and after the interventions. Several analyses were performed to gain better insight into the mechanisms by which muscle function was improved. We determined cumulative rates of myofibrillar and mitochondrial protein synthesis using 2H2O labelling and mass spectrometry; assessed ex vivo contractile properties and in vitro mitochondrial function, evaluated neuromuscular junction (NMJ) stability, and assessed implicated molecular singling pathways. Furthermore, whole-body and muscle insulin sensitivity along with glucose metabolism, were evaluated using a hyperinsulinaemic-euglycaemic clamp. RESULTS: EAA + RET increased muscle mass (10%, P < 0.05) and strength (6%, P < 0.05) more than RET alone, due to an enhanced rate of integrated muscle protein synthesis (19%, P < 0.05) with concomitant activation of Akt1/mTORC1 signalling. Muscle quality (muscle strength normalized to mass) was improved by RET (i.e., RET and EAA + RET) compared with sedentary groups (10%, P < 0.05), which was associated with increased AchR cluster size and MuSK activation (P < 0.05). EAA + RET also increased endurance capacity more than RET alone (26%, P < 0.05) by increasing both mitochondrial protein synthesis (53%, P < 0.05) and DRP1 activation (P < 0.05). Maximal respiratory capacity increased (P < 0.05) through activation of the mTORC1-DRP1 signalling axis. These favourable effects were accompanied by an improvement in basal glucose metabolism (i.e., blood glucose concentrations and endogenous glucose production vs. CON, P < 0.05). CONCLUSIONS: Combined treatment with balanced free EAAs and RET may effectively promote endurance capacity as well as muscle strength through increased muscle protein synthesis, improved NMJ stability, and enhanced mitochondrial dynamics via mTORC1-DRP1 axis activation, ultimately leading to improved basal glucose metabolism.

Overexpression of UCP3 decreases mitochondrial efficiency in mouse skeletal muscle in vivo.

Uncoupling protein-3 (UCP3) is a mitochondrial transmembrane protein highly expressed in the muscle that has been implicated in regulating the efficiency of mitochondrial oxidative phosphorylation. Increasing UCP3 expression in skeletal muscle enhances proton leak across the inner mitochondrial membrane and increases oxygen consumption in isolated mitochondria, but its precise function in vivo has yet to be fully elucidated. To examine whether muscle-specific overexpression of UCP3 modulates muscle mitochondrial oxidation in vivo, rates of ATP synthesis were assessed by 31 P magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-13 C]acetate incorporation into muscle [4-13 C]-, [3-13 C]-glutamate, and [4-13 C]-glutamine by high-resolution 13 C/1 H MRS. Using this approach, we found that the overexpression of UCP3 in skeletal muscle was accompanied by increased muscle mitochondrial inefficiency in vivo as reflected by a 42% reduction in the ratio of ATP synthesis to mitochondrial oxidation.

Polyphenolic Compounds Isolated from Marine Algae Attenuate the Replication of SARS-CoV-2 in the Host Cell through a Multi-Target Approach of 3CLpro and PLpro.

A global health concern has emerged as a response to the recent SARS-CoV-2 pandemic. The identification and inhibition of drug targets of SARS-CoV-2 is a decisive obligation of scientists. In addition to the cell entry mechanism, SARS-CoV-2 expresses a complicated replication mechanism that provides excellent drug targets. Papain-like protease (PLpro) and 3-chymotrypsin-like protease (3CLpro) play a vital role in polyprotein processing, producing functional non-structural proteins essential for viral replication and survival in the host cell. Moreover, PLpro is employed by SARS-CoV-2 for reversing host immune responses. Therefore, if some particular compound has the potential to interfere with the proteolytic activities of 3CLpro and PLpro of SARS-CoV-2, it may be effective as a treatment or prophylaxis for COVID-19, reducing viral load, and reinstating innate immune responses. Thus, the present study aims to inhibit SARS-CoV-2 through 3CLpro and PLpro using marine natural products isolated from marine algae that contain numerous beneficial biological activities. Molecular docking analysis was utilized in the present study for the initial screening of selected natural products depending on their 3CLpro and PLpro structures. Based on this approach, Ishophloroglucin A (IPA), Dieckol, Eckmaxol, and Diphlorethohydroxycarmalol (DPHC) were isolated and used to perform in vitro evaluations. IPA presented remarkable inhibitory activity against interesting drug targets. Moreover, Dieckol, Eckmaxol, and DPHC also expressed significant potential as inhibitors. Finally, the results of the present study confirm the potential of IPA, Dieckol, Eckmaxol, and DPHC as inhibitors of SARS-CoV-2. To the best of our knowledge, this is the first study that assesses the use of marine natural products as a multifactorial approach against 3CLpro and PLpro of SARS-CoV-2.

Self-transducible LRS-UNE-L peptide enhances muscle regeneration.

BACKGROUND: Muscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C-terminal unique attached sequence motif (UNE) domain of leucyl-tRNA synthetase (LRS-UNE-L) as an mTORC1 (mTOR complex1)-activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle-enhancing peptide. METHODS: In vitro Vps34 lipid kinase assay, phosphatidylinositol 3-phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS-UNE-L on the Vps34-PLD1-mTOR pathway. Adeno-associated virus (AAV)-LRS-UNE-L was transduced in C2C12 cells in vitro, in BaCl2 -injured tibialis anterior (TA) muscles, and in 18-month-old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle-specific cell-permeable peptide M12 was fused with LRS-UNE-L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12-LRS-UNE-L was introduced into BaCl2 -injured TA muscles of 15-week-old Pld1+/+ or Pld1-/- mice, and its effect was analysed by measurement of cross-sectional area of regenerating muscle fibres. RESULTS: The LRS-UNE-L expression restored amino acid-induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases-independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS-UNE-L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid-treated control cells, but it did not affect autophagic flux. AAV-delivered LRS-UNE-L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin-like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV-LRS-UNE-L increased the size of regenerating muscle fibres in BaCl2 -injured TA muscles (124%, P = 0.0279) (n = 9-10), but it did not change the muscle fibre size of TA muscles in old mice. M12-LRS-UNE-L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl2 -injured TA muscles in a PLD1-dependent manner (116%, P = 0.0022) (n = 6). CONCLUSIONS: Our results provide compelling evidence that M12-LRS-UNE-L could be a muscle-enhancing protein targeting mTOR.

Bone morphogenic protein 9 is a novel thermogenic hepatokine secreted in response to cold exposure.

OBJECTIVE: Maintaining a constant core body temperature is essential to homeothermic vertebrate survival. Adaptive thermogenesis in brown adipose tissue and skeletal muscle is the primary mechanism of adjustment to an external stimulus such as cold exposure. Recently, several reports have revealed that the liver can play a role as a metabolic hub during adaptive thermogenesis. In this study, we suggest that the liver plays a novel role in secreting thermogenic factors in adaptive thermogenesis. Bone morphogenetic protein 9 (BMP9) is a hepatokine that regulates many biological processes, including osteogenesis, chondrogenesis, hematopoiesis, and angiogenesis. Previously, BMP9 was suggested to affect preadipocyte proliferation and differentiation. However, the conditions and mechanisms underlying hepatic expression and secretion and adipose tissue browning of BMP9 remain largely unknown. In this study, we investigated the physiological conditions for secretion and the regulatory mechanism of hepatic Bmp9 expression and the molecular mechanism by which BMP9 induces thermogenic gene program activation in adipose tissue. Here, we also present the pharmacological effects of BMP9 on a high-fat-induced obese mouse model. METHODS: To investigate the adaptive thermogenic role of BMP9 in vivo, we challenged mice with cold temperature exposure for 3 weeks and then examined the BMP9 plasma concentration and hepatic expression level. The cellular mechanism of hepatic Bmp9 expression under cold exposure was explored through promoter analysis. To identify the role of BMP9 in the differentiation of brown and beige adipocytes, we treated pluripotent stem cells and inguinal white adipose tissue (iWAT)-derived stromal-vascular (SV) cells with BMP9, and brown adipogenesis was monitored by examining thermogenic gene expression and signaling pathways. Furthermore, to evaluate the effect of BMP9 on diet-induced obesity, changes in body composition and glucose tolerance were analyzed in mice administered recombinant BMP9 (rBMP9) for 8 weeks. RESULTS: Hepatic Bmp9 expression and plasma levels in mice were significantly increased after 3 weeks of cold exposure. Bmp9 mRNA expression in the liver was regulated by transcriptional activation induced by cAMP response-element binding protein (CREB) and CREB-binding protein (CBP) on the Bmp9 promoter. Treatment with BMP9 promoted the differentiation of multipotent stem cells and iWAT-derived SV cells into beige adipocytes, as indicated by the increased expression of brown adipocyte and mitochondrial biogenesis markers. Notably, activation of the mothers against decapentaplegic homolog 1 (Smad1) and p44/p42 mitogen-activated protein kinase (MAPK) pathways was required for the induction of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression in BMP9-induced differentiation of SVs into beige adipocytes. The administration of rBMP9 in vivo also induced browning markers in white adipose tissue. In high-fat diet-induced obese mice, rBMP9 administration conferred protection against obesity and enhanced glucose tolerance. CONCLUSIONS: BMP9 is a hepatokine regulated by cold-activated CREB and CBP and enhances glucose and fat metabolism by promoting the activation of the thermogenic gene program in adipocytes. These data implicate BMP9 as a potential pharmacological tool for protecting against obesity and type 2 diabetes.

Essential Amino Acid-Enriched Diet Alleviates Dexamethasone-Induced Loss of Muscle Mass and Function through Stimulation of Myofibrillar Protein Synthesis and Improves Glucose Metabolism in Mice.

Dexamethasone (DEX) induces dysregulation of protein turnover, leading to muscle atrophy and impairment of glucose metabolism. Positive protein balance, i.e., rate of protein synthesis exceeding rate of protein degradation, can be induced by dietary essential amino acids (EAAs). In this study, we investigated the roles of an EAA-enriched diet in the regulation of muscle proteostasis and its impact on glucose metabolism in the DEX-induced muscle atrophy model. Mice were fed normal chow or EAA-enriched chow and were given daily injections of DEX over 10 days. We determined muscle mass and functions using treadmill running and ladder climbing exercises, protein kinetics using the D2O labeling method, molecular signaling using immunoblot analysis, and glucose metabolism using a U-13C6 glucose tracer during oral glucose tolerance test (OGTT). The EAA-enriched diet increased muscle mass, strength, and myofibrillar protein synthesis rate, concurrent with improved glucose metabolism (i.e., reduced plasma insulin concentrations and increased insulin sensitivity) during the OGTT. The U-13C6 glucose tracing revealed that the EAA-enriched diet increased glucose uptake and subsequent glycolytic flux. In sum, our results demonstrate a vital role for the EAA-enriched diet in alleviating the DEX-induced muscle atrophy through stimulation of myofibrillar proteins synthesis, which was associated with improved glucose metabolism.

Insulin Resistance: From Mechanisms to Therapeutic Strategies.

Insulin resistance is the pivotal pathogenic component of many metabolic diseases, including type 2 diabetes mellitus, and is defined as a state of reduced responsiveness of insulin-targeting tissues to physiological levels of insulin. Although the underlying mechanism of insulin resistance is not fully understood, several credible theories have been proposed. In this review, we summarize the functions of insulin in glucose metabolism in typical metabolic tissues and describe the mechanisms proposed to underlie insulin resistance, that is, ectopic lipid accumulation in liver and skeletal muscle, endoplasmic reticulum stress, and inflammation. In addition, we suggest potential therapeutic strategies for addressing insulin resistance.

Anti-Inflammatory Mechanisms of Fucoidans to Treat Inflammatory Diseases: A Review.

Fucoidans are sulfated heteropolysaccharides found in the cell walls of brown seaweeds (Phaeophyceae) and in some marine invertebrates. Generally, fucoidans are composed of significant amounts of L-fucose and sulfate groups, and lesser amounts of arabinose, galactose, glucose, glucuronic acid, mannose, rhamnose, and xylose. In recent years, fucoidans isolated from brown seaweeds have gained considerable attention owing to their promising bioactive properties such as antioxidant, immunomodulatory, anti-inflammatory, antiobesity, antidiabetic, and anticancer properties. Inflammation is a complex immune response that protects the organs from infection and tissue injury. While controlled inflammatory responses are beneficial to the host, leading to the removal of immunostimulants from the host tissues and restoration of structural and physiological functions in the host tissues, chronic inflammatory responses are often associated with the pathogenesis of tumor development, arthritis, cardiovascular diseases, diabetes, obesity, and neurodegenerative diseases. In this review, the authors mainly discuss the studies since 2016 that have reported anti-inflammatory properties of fucoidans isolated from various brown seaweeds, and their potential as a novel functional material for the treatment of inflammatory diseases.

Ablation of USP21 in skeletal muscle promotes oxidative fibre phenotype, inhibiting obesity and type 2 diabetes.

BACKGROUND: Skeletal muscle as a metabolic consumer determines systemic energy homeostasis by regulating myofibre type conversion and muscle mass control. Perturbation of the skeletal muscle metabolism elevates the risk of a variety of diseases including metabolic disorders. However, the regulatory pathways and molecules are not completely understood. The discovery of relevant responsible molecules and the associated network could be an attractive strategy to overcome diseases associated with muscle problems. METHODS: An initial screening using quantitative trait locus analysis enabled us to extract a set of genes including ubiquitin-specific proteases21 (USP21) (r = 0.738; P = 0.004) as potential targets associated with fasting blood glucose content. Given tight regulation of the ubiquitination status of proteins in muscle, we focused on USP21 and generated whole-body (KO) and skeletal muscle-specific USP21 knockout (MKO) mice. Transcriptomics, proteomics, and lipidomics assays in combination with various in vivo and in vitro experiments were performed to understand the functions of USP21 and underlying mechanisms. A high-fat diet (60%)-fed mouse model and diabetic patient-derived samples were utilized to assess the effects of USP21 on energy metabolism in skeletal muscle. RESULTS: USP21 was highly expressed in both human and mouse skeletal muscle, and controlled skeletal muscle oxidative capacity and fuel consumption. USP21-KO or USP21-MKO significantly promoted oxidative fibre type changes (Δ36.6% or Δ47.2%), muscle mass increase (Δ13.8% to Δ22.8%), and energy expenditure through mitochondrial biogenesis, fatty acid oxidation, and UCP2/3 induction (P < 0.05 or P < 0.01). Consistently, cold exposure repressed USP21 expression in mouse skeletal muscle (Δ55.3%), whereas loss of USP21 increased thermogenesis (+1.37°C or +0.84°C; P < 0.01). Mechanistically, USP21 deubiquitinated DNA-PKcs and ACLY, which led to AMPK inhibition. Consequently, USP21 ablation diminished diet-induced obesity (WT vs. USP21-KO, Δ8.02 g, 17.1%, P < 0.01; litter vs. USP21-MKO, Δ3.48 g, 7.7%, P < 0.05) and insulin resistance. These findings were corroborated in a skeletal muscle-specific gene KO mouse model. USP21 was induced in skeletal muscle of a diabetic patient (1.94-fold), which was reciprocally changed to p-AMPK (0.30-fold). CONCLUSIONS: The outcomes of this research provide novel information as to how USP21 in skeletal muscle contributes to systemic energy homeostasis, demonstrating USP21 as a key molecule in the regulation of myofibre type switch, muscle mass control, mitochondrial function, and heat generation and, thus, implicating the potential of this molecule and its downstream substrates network as targets for the treatment and/or prevention of muscle dysfunction and the associated metabolic diseases.

Myostatin Inhibition-Induced Increase in Muscle Mass and Strength Was Amplified by Resistance Exercise Training, and Dietary Essential Amino Acids Improved Muscle Quality in Mice.

It has been frequently reported that myostatin inhibition increases muscle mass, but decreases muscle quality (i.e., strength/muscle mass). Resistance exercise training (RT) and essential amino acids (EAAs) are potent anabolic stimuli that synergistically increase muscle mass through changes in muscle protein turnover. In addition, EAAs are known to stimulate mitochondrial biogenesis. We have investigated if RT amplifies the anabolic potential of myostatin inhibition while EAAs enhance muscle quality through stimulations of mitochondrial biogenesis and/or muscle protein turnover. Mice were assigned into ACV (myostatin inhibitor), ACV+EAA, ACV+RT, ACV+EAA +RT, or control (CON) over 4 weeks. RT, but not EAA, increased muscle mass above ACV. Despite differences in muscle mass gain, myofibrillar protein synthesis was stimulated similarly in all vs. CON, suggesting a role for changes in protein breakdown in muscle mass gains. There were increases in MyoD expression but decreases in Atrogin-1/MAFbx expression in ACV+EAA, ACV+RT, and ACV+EAA+RT vs. CON. EAA increased muscle quality (e.g., grip strength and maximal carrying load) without corresponding changes in markers of mitochondrial biogenesis and neuromuscular junction stability. In conclusion, RT amplifies muscle mass and strength through changes in muscle protein turnover in conjunction with changes in implicated signaling, while EAAs enhance muscle quality through unknown mechanisms.

TFEB-GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response.

TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca2+-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB-GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB-GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.

Mitohormesis in Hypothalamic POMC Neurons Mediates Regular Exercise-Induced High-Turnover Metabolism.

Low-grade mitochondrial stress can promote health and longevity, a phenomenon termed mitohormesis. Here, we demonstrate the opposing metabolic effects of low-level and high-level mitochondrial ribosomal (mitoribosomal) stress in hypothalamic proopiomelanocortin (POMC) neurons. POMC neuron-specific severe mitoribosomal stress due to Crif1 homodeficiency causes obesity in mice. By contrast, mild mitoribosomal stress caused by Crif1 heterodeficiency in POMC neurons leads to high-turnover metabolism and resistance to obesity. These metabolic benefits are mediated by enhanced thermogenesis and mitochondrial unfolded protein responses (UPRmt) in distal adipose tissues. In POMC neurons, partial Crif1 deficiency increases the expression of ß-endorphin (ß-END) and mitochondrial DNA-encoded peptide MOTS-c. Central administration of MOTS-c or ß-END recapitulates the adipose phenotype of Crif1 heterodeficient mice, suggesting these factors as potential mediators. Consistently, regular running exercise at moderate intensity stimulates hypothalamic MOTS-c/ß-END expression and induces adipose tissue UPRmt and thermogenesis. Our findings indicate that POMC neuronal mitohormesis may underlie exercise-induced high-turnover metabolism.

Magnetic Resonance-Based Assessments Better Capture Pathophysiologic Profiles and Progression in Nonalcoholic Fatty Liver Disease.

BACKGROUND: Several noninvasive tools are available for the assessment of nonalcoholic fatty liver disease (NAFLD) including clinical and blood biomarkers, transient elastography (TE), and magnetic resonance imaging (MRI) techniques, such as proton density fat fraction (MRI-PDFF) and magnetic resonance elastography (MRE). In the present study, we aimed to evaluate whether magnetic resonance (MR)-based examinations better discriminate the pathophysiologic features and fibrosis progression in NAFLD than other noninvasive methods. METHODS: A total of 133 subjects (31 healthy volunteers and 102 patients with NAFLD) were subjected to clinical and noninvasive NAFLD evaluation, with additional liver biopsy in some patients (n=54). RESULTS: MRI-PDFF correlated far better with hepatic fat measured by MR spectroscopy (r=0.978, P<0.001) than with the TE controlled attenuation parameter (CAP) (r=0.727, P<0.001). In addition, MRI-PDFF showed stronger correlations with various pathophysiologic parameters for cellular injury, glucose and lipid metabolism, and inflammation, than the TE-CAP. The MRI-PDFF and TE-CAP cutoff levels associated with abnormal elevation of serum alanine aminotransferase were 9.9% and 270 dB/m, respectively. The MRE liver stiffness measurement (LSM) showed stronger correlations with liver enzymes, platelets, complement component 3, several clinical fibrosis scores, and the enhanced liver fibrosis (ELF) score than the TE-LSM. In an analysis of only biopsied patients, MRE performed better in discriminating advanced fibrosis with a cutoff value of 3.9 kPa than the TE (cutoff 8.1 kPa) and ELF test (cutoff 9.2 kPa). CONCLUSION: Our results suggest that MRI-based assessment of NAFLD is the best non-invasive tool that captures the histologic, pathophysiologic and metabolic features of the disease.

Prokineticin receptor 1 ameliorates insulin resistance in skeletal muscle.

Type 2 diabetes mellitus may result from insulin resistance in skeletal muscle. Prokineticin receptor 1 (Prokr1) improves metabolic phenotype in adipose tissue and the cardiovascular system; however, its effects on skeletal muscle have not been investigated. We investigated the Prokr1 signaling pathways and its metabolic function in murine myoblast, satellite cells, and their differentiated myotubes. We measured the expression levels of Prokr1 in the skeletal muscle of mice as well as human skeletal muscle cell-derived myotubes. Prokineticin 2 (PROK2), a ligand of PROKR1, induced calcium mobilization in a dose-dependent manner and altered the mRNA levels of 578 genes in PROKR1-overexpressed HEK293T cells. Functional enrichment of differentially expressed genes revealed that PROKR1 activated Gq-mediated PI3K/AKT and MAPK/ERK signaling pathways in skeletal muscle cells. Prokr1 significantly activated the PI3K/AKT signaling pathway in myotubes derived from C2C12 and satellite cells, regardless of the presence or absence of insulin. Prokr1 also promoted the translocation of glucose transporter 4 (GLUT4) into the plasma membrane. In palmitate-induced insulin-resistant myotubes, Prokr1 enhanced insulin-stimulated AKT phosphorylation, GLUT4 translocation, and glucose uptake. mRNA and protein levels of Prokr1 were significantly decreased in skeletal muscle and white adipose tissue of diet-induced obese mice, and the amount of PROKR1 protein was significantly decreased in human skeletal muscle cell-derived myotubes under insulin resistance conditions. Taken together, these results demonstrate that Prokr1 plays an important role in insulin sensitivity and is a potential therapeutic target to ameliorate insulin resistance in skeletal muscle.

Prevention of Oxidative Stress-Induced Pancreatic Beta Cell Damage by Broussonetia Kazinoki Siebold Fruit Extract Via the ERK-Nox4 Pathway.

Pancreatic beta cells are vulnerable to oxidative stress, which causes beta cell death and dysfunction in diabetes mellitus. Broussonetia kazinoki Siebold (BK) is a widely used herbal medicine, but its potential effects against beta cell death-induced diabetes have not been studied. Therefore, we investigated the protective effect of an ethanolic extract of BK fruit (BKFE) against streptozotocin (STZ)-induced toxicity in pancreatic beta cells. Intraperitoneal injection of STZ in mice induced hyperglycemia; however, oral administration of BKFE significantly decreased the blood glucose level as well as HbA1c levels. BKFE treatment improved glucose tolerance and increased body weight in diabetic mice. Moreover, BKFE treatment resulted in increased serum insulin levels and insulin expression in the pancreas as well as decreased 4-hydroxynonenal levels induced by oxidative stress. Treatment with STZ decreased cell viability of mouse insulinoma cells (MIN6), which was blocked by BKFE pretreatment. BKFE significantly inhibited apoptotic cells and decreased the expression levels of cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase (PARP) induced by STZ treatment. Production of reactive oxygen species in STZ-treated MIN6 cells was also significantly decreased by treatment with BKFE. Erk phosphorylation and Nox4 levels increased in STZ-treated MIN6 cells and the pancreas of mice injected with STZ and this increase was inhibited by treatment with BKFE. Inhibition of Erk phosphorylation by treatment with the PD98059 inhibitor or siRNA Erk also blocked the expression of Nox4 induced by STZ treatment. In conclusion, BKFE inhibits Erk phosphorylation, which in turn prevents STZ-induced oxidative stress and beta cell apoptosis. These results suggested that BKFE can be used to prevent or treat beta cell damage in diabetes.

The essential role of fructose-1,6-bisphosphatase 2 enzyme in thermal homeostasis upon cold stress.

Skeletal muscle is a major organ for glucose disposal and thermogenesis. While hepatic fructose-1,6-bisphosphatase is well known as a key enzyme for gluconeogenesis, the role of muscle fructose-1,6-bisphosphatase 2 (Fbp2) in glucose disposal and thermogenesis is unknown. Here, using Fbp2 knockout (KO) mice, we assessed the physiological role of Fbp2 in energy and glucose metabolism and thermogenesis. In vivo assessments of energy metabolism, glucose metabolism, and thermogenesis were performed by indirect calorimetry, hyperinsulinemic-euglycemic clamp, and cold challenge studies, respectively. Under both feeding and fasting conditions, Fbp2 KO mice showed similar phenotypes regarding energy and glucose metabolism compared to wild-type (WT) mice. However, Fbp2 KO mice were severely intolerant to cold challenge under fasting conditions. Mechanistically, the cold-induced intramuscular conversion of lactate to glycogen (glyconeogenesis) is completely abolished in the KO muscle, which leads to a lack of glycogen source for thermogenesis in Fbp2 KO mice. The cold-intolerant phenotype of KO mice disappeared after feeding, and the KO mice were equally as cold tolerant as the WT mice and survived during the cold challenge for three weeks. Taken together, these data demonstrate that Fbp2 is essential for muscle thermogenesis by replenishing the intramuscular glycogen pool through glyconeogenesis when the exogenous glucose source is limited. These data imply the physiological importance of Fbp2 in thermal homeostasis and suggest a potential novel therapy targeted to increase glycogen replenishment upon cold stress.

Grasp55-/- mice display impaired fat absorption and resistance to high-fat diet-induced obesity.

The Golgi apparatus plays a central role in the intracellular transport of macromolecules. However, molecular mechanisms of Golgi-mediated lipid transport remain poorly understood. Here, we show that genetic inactivation of the Golgi-resident protein GRASP55 in mice reduces whole-body fat mass via impaired intestinal fat absorption and evokes resistance to high-fat diet induced body weight gain. Mechanistic analyses reveal that GRASP55 participates in the Golgi-mediated lipid droplet (LD) targeting of some LD-associated lipases, such as ATGL and MGL, which is required for sustained lipid supply for chylomicron assembly and secretion. Consequently, GRASP55 deficiency leads to reduced chylomicron secretion and abnormally large LD formation in intestinal epithelial cells upon exogenous lipid challenge. Notably, deletion of dGrasp in Drosophila causes similar defects of lipid accumulation in the midgut. These results highlight the importance of the Golgi complex in cellular lipid regulation, which is evolutionary conserved, and uncover potential therapeutic targets for obesity-associated diseases.

Transcriptional coactivator with PDZ-binding motif suppresses the expression of steroidogenic enzymes by nuclear receptor 4 A1 in Leydig cells.

Transcriptional coactivator with PDZ-binding motif (TAZ) plays crucial role in maintaining testicular structure and function via regulation of senescence of spermatogenic cells. However, it remains unclear whether TAZ is involved in testosterone biosynthesis in testicular Leydig cells. We found that TAZ deficiency caused aberrant Leydig cell expansion and increased lipid droplet formation, which was significantly associated with increased lipogenic enzyme expression. Additionally, the expression of key steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 (CYP) 11A1, CYP17A1, and 3ß-hydroxysteroid dehydrogenase, was greatly increased in TAZ-deficient testes and primary Leydig cells. Interestingly, the transcriptional activity of nuclear receptor 4 A1 (NR4A1) was dramatically suppressed by TAZ; however, the protein expression and the subcellular localization of NR4A1 were not affected by TAZ. TAZ directly associated with the N-terminal region of NR4A1 and substantially suppressed its DNA-binding and transcriptional activities. Stable expression of TAZ in the mouse Leydig TM3 cell line decreased the expression of key steroidogenic enzymes, whereas knockdown of endogenous TAZ in TM3 cells increased transcripts of steroidogenic genes induced by NR4A1. Consistently, testosterone production was enhanced within TAZ-deficient Leydig cells. However, TAZ deficiency resulted in decreased testosterone secretion caused by dysfunctional mitochondria and lysosomes. Therefore, TAZ plays essential role in NR4A1-induced steroidogenic enzyme expression and testosterone production in Leydig cells.

Exogenous 8-hydroxydeoxyguanosine ameliorates liver fibrosis through the inhibition of Rac1-NADPH oxidase signaling.

BACKGROUND AND AIM: Exogenous 8-hydroxydeoxyguanosine (8-OHdG) was suggested as an inhibitor of Rac1 and NADPH oxidase (NOX). The aim of this study was to evaluate the effects of the exogenous 8-OHdG on hepatic fibrogenesis in vitro and in vivo model of liver fibrosis. METHODS: Adult Sprague-Dawley rats were allocated to sham-operated rats (n = 7), rats that underwent bile duct ligation (BDL) (n = 6), and BDL rats treated with 8-OHdG (60 mg/kg/day by gavage, n = 6). All rats were sacrificed on day 21. Double immunofluorescence staining between either NOX1 or NOX2 and α-smooth muscle actin (SMA) in liver was performed. Hepatic fibrotic contents were assessed by hydroxyproline assay and quantified by Sirius red staining. In vitro, hepatic stellate cell (HSC) line LX-2 and HHSteC cells were stimulated by angiotensin II (10 µM). The reactive oxygen species (ROS) production was measured by confocal microscopy. The expressions of NOX1, NOX2, α-SMA, transforming growth factor (TGF)-ß1, and collagen Iα were analyzed by quantitative real-time polymerase chain reaction or immunoblotting. RESULTS: The 8-OHdG treatment in BDL rats reduced the NOX1 and NOX2 protein expression, which overlapped with α-SMA compared with BDL rats. The 8-OHdG treatment in BDL rats significantly decreased the mRNA expression of NOX1, NOX2, α-SMA, TGF-ß1, and collagen Iα, and fibrotic contents. Increases of ROS production, Rac1 activation, NOX1, NOX2, and fibronectin expression induced by angiotensin II in HSCs were attenuated by 8-OHdG. CONCLUSIONS: Rac1 activation and NOX-derived ROS are implicated to liver fibrosis. The 8-OHdG ameliorates liver fibrosis through the inhibition of Rac1 activation and NOX-derived ROS.

An inverse agonist of estrogen-related receptor γ regulates 2-arachidonoylglycerol synthesis by modulating diacylglycerol lipase expression in alcohol-intoxicated mice.

Chronic alcohol feeding increases the levels of 2-arachidonoylglycerol (2-AG) in the liver, which activates hepatic cannabinoid receptor type 1 (CB1R), leading to oxidative liver injury. 2-AG biosynthesis is catalyzed by diacylglycerol lipase (DAGL). However, the mechanisms regulating hepatic DAGL gene expression and 2-AG production are largely unknown. In this study, we show that CB1R-induced estrogen-related receptor γ (ERRγ) controls hepatic DAGL gene expression and 2-AG levels. Arachidonyl-2'-chloroethylamide (ACEA), a synthetic CB1R agonist, significantly upregulated ERRγ, DAGLα, and DAGLß, and increased 2-AG levels in the liver (10 mg/kg) and hepatocytes (10 µM) of wild-type (WT) mice. ERRγ overexpression upregulated DAGLα and DAGLß expressions and increased 2-AG levels, whereas ERRγ knockdown abolished ACEA-induced DAGLα, DAGLß, and 2-AG in vitro and in vivo. Promoter assays showed that ERRγ positively regulated DAGLα and DAGLß transcription by binding to the ERR response element in the DAGLα and DAGLß promoters. Chronic alcohol feeding (27.5% of total calories) induced hepatic steatosis and upregulated ERRγ, leading to increased DAGLα, DAGLß, or 2-AG in WT mice, whereas these alcohol-induced effects did not occur in hepatocyte-specific CB1R knockout mice or in those treated with the ERRγ inverse agonist GSK5182 (40 mg/kg in mice and 10 µM in vitro). Taken together, these results indicate that suppression of alcohol-induced DAGLα and DAGLß gene expressions and 2-AG levels by an ERRγ-specific inverse agonist may be a novel and attractive therapeutic approach for the treatment of alcoholic liver disease.