A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice.
ABSTRACT: As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.
Project description:Dissecting exercise-mimicking pathways that can replicate the benefits of exercise in obesity and diabetes may lead to promising treatments for metabolic disorders. Muscle estrogen-related receptor gamma (ERR?) is induced by exercise, and when over-expressed in the skeletal muscle mimics exercise by stimulating glycolytic-to-oxidative myofiber switch, mitochondrial biogenesis and angiogenesis in lean mice. The objective of this study was to test whether muscle ERR? in obese mice mitigates weight gain and insulin resistance. To do so, ERR? was selectively over-expressed in the skeletal muscle of obese and diabetic db/db mice. Muscle ERR? over-expression successfully triggered glycolytic-to-oxidative myofiber switch, increased functional mitochondrial content and boosted vascular supply in the db/db mice. Despite aerobic remodeling, ERR? surprisingly failed to improve whole-body energy expenditure, block muscle accumulation of triglycerides, toxic diacylglycerols (DAG) and ceramides or suppress muscle PKC? sarcolemmal translocation in db/db mice. Consequently, muscle ERR? did not mitigate impaired muscle insulin signaling or insulin resistance in these mice. In conclusion, obesity and diabetes in db/db mice are not amenable to selective ERR?-directed programming of classic exercise-like effects in the skeletal muscle. Other biochemical pathways or integrated whole-body effects of exercise may be critical for resisting diabetes and obesity.
Project description:To identify metabolic derangements contributing to diabetes susceptibility in the leptin receptor-deficient obese C57BLKS/J-db/db (BKS-db) mouse strain.Young BKS-db mice were used to identify metabolic pathways contributing to the development of diabetes. Using the diabetes-resistant B6-db strain as a comparison, in vivo and in vitro approaches were applied to identify metabolic and molecular differences between the two strains.Despite higher plasma insulin levels, BKS-db mice exhibit lower lipogenic gene expression, rate of lipogenesis, hepatic triglyceride and glycogen content, and impaired insulin suppression of gluconeogenic genes. Hepatic insulin receptor substrate (IRS)-1 and IRS-2 expression and insulin-stimulated Akt-phosphorylation are decreased in BKS-db primary hepatocytes. Hyperinsulinemic-euglycemic clamp studies indicate that in contrast to hepatic insulin resistance, skeletal muscle is more insulin sensitive in BKS-db than in B6-db mice. We also demonstrate that elevated plasma triglyceride levels in BKS-db mice are associated with reduced triglyceride clearance due to lower lipase activities.Our study demonstrates the presence of metabolic derangements in BKS-db before the onset of beta-cell failure and identifies early hepatic insulin resistance as a component of the BKS-db phenotype. We propose that defects in hepatic insulin signaling contribute to the development of diabetes in the BKS-db mouse strain.
Project description:Peroxisome proliferator-activated receptor-? coactivator-1? (PGC-1?) has been shown to influence energy metabolism. Hence, we explored a strategy to target PGC-1? expression to treat metabolic syndromes. We developed a high-throughput screening assay that uses the human PGC-1? promoter to drive expression of luciferase. The effects of lead compound stimulation on PGC-1? expression in muscle cells and hepatocytes were investigated in vitro and in vivo. A novel small molecule, ZLN005, led to changes in PGC-1? mRNA levels, glucose uptake, and fatty acid oxidation in L6 myotubes. Activation of AMP-activated protein kinase was involved in the induction of PGC-1? expression. In diabetic db/db mice, chronic administration of ZLN005 increased PGC-1? and downstream gene transcription in skeletal muscle, whereas hepatic PGC-1? and gluconeogenesis genes were reduced. ZLN005 increased fat oxidation and improved the glucose tolerance, pyruvate tolerance, and insulin sensitivity of diabetic db/db mice. Hyperglycemia and dyslipidemia also were ameliorated after treatment with ZLN005. Our results demonstrated that a novel small molecule selectively elevated the expression of PGC-1? in myotubes and skeletal muscle and exerted promising therapeutic effects for treating type 2 diabetes.
Project description:Muscle atrophy occurs in many pathological states, including cancer, diabetes and sepsis, whose results primarily from accelerated protein degradation and activation of the ubiquitin-proteasome pathway. Expression of Muscle RING finger 1 (MuRF1), an E3 ubiquitin ligase, was increased to induce the loss of muscle mass in diabetic condition. However, hydrogen sulphide (H2 S) plays a crucial role in the variety of physiological functions, including antihypertension, antiproliferation and antioxidant. In this study, db/db mice and C2C12 myoblasts treated by high glucose and palmitate and oleate were chose as animal and cellular models. We explored how exogenous H2 S attenuated the degradation of skeletal muscle via the modification of MuRF1 S-sulfhydration in db/db mice. Our results show cystathionine-r-lyase expression, and H2 S level in skeletal muscle of db/db mice was reduced. Simultaneously, exogenous H2 S could alleviate ROS production and reverse expression of ER stress protein markers. Exogenous H2 S could decrease the ubiquitination level of MYOM1 and MYH4 in db/db mice. In addition, exogenous H2 S reduced the interaction between MuRF1 with MYOM1 and MYH4 via MuRF1 S-sulfhydration. Based on these results, we establish that H2 S prevented the degradation of skeletal muscle via MuRF1 S-sulfhydration at the site of Cys44 in db/db mice.
Project description:Activation of aldosterone/mineralocorticoid receptors (MR) has been implicated in vascular dysfunction of diabetes. Underlying mechanisms are elusive. Therefore, we investigated the role of Rho kinase (ROCK) in aldosterone/MR signaling and vascular dysfunction in a model of diabetes. Diabetic obese mice (db/db) and control counterparts (db/+) were treated with MR antagonist (MRA, potassium canrenoate, 30?mg/kg/day, 4 weeks) or ROCK inhibitor, fasudil (30?mg/kg/day, 3 weeks). Plasma aldosterone was increased in db/db versus db/+. This was associated with enhanced vascular MR signaling. Norepinephrine (NE)-induced contraction was increased in arteries from db/db mice. These responses were attenuated in mice treated with canrenoate or fasudil. Db/db mice displayed hypertrophic remodeling and increased arterial stiffness, improved by MR blockade. Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Vascular hypercontractility in db/db mice was associated with increased myosin light chain phosphorylation and reduced expression of PKG-1?. Vascular RhoA/ROCK signaling and expression of pro-inflammatory and pro-fibrotic markers were exaggerated in db/db mice, effects that were attenuated by MRA. Fasudil, but not MRA, improved vascular insulin sensitivity in db/db mice, evidenced by normalization of Irs1 phosphorylation. Our data identify novel pathways involving MR-RhoA/ROCK-PKG-1 that underlie vascular dysfunction and injury in diabetic mice.
Project description:The mechanism by which adipocyte-derived endocrine factors promote insulin resistance in skeletal muscle are not fully understood. MiR-27a is highly expressed in sera of obese individuals with prediabetes and T2DM, and mainly derived by adipose tissues. Thus, miR-27a secreted into circulation by adipose tissue may regulate insulin resistance in skeletal muscle. Methods: The association between miR-27a and insulin resistance in skeletal muscle was determined in obese children, high-fat diet-induced miR-27a knockdown obese mice, db/db mice and C2C12 cells overexpressing miR-27a. The crosstalk mediated by exosomal miR-27a between adipose tissue and skeletal muscle was determined in C2C12 cells incubated with conditioned medium prepared from palmitate-treated 3T3-L1 adipocytes. Results: We showed that serum miR-27a level correlated positively with obesity and insulin resistance in obese children, and that elevated serum miR-27a levels correlated with insulin resistance in leptin receptor-deficient db/db mice, and with obesity and insulin resistance in high-fat diet-fed C57BL/6J mice. MiR-27a released from adipocytes of high-fat diet-fed C57BL/6J mice was associated with triglyceride accumulation. MiR-27a derived from these adipocytes induced insulin resistance in C2C12 skeletal muscle cells through miR-27a-mediated repression of PPAR? and its downstream genes involved in the development of obesity. Conclusions: These results identify a novel crosstalk signaling pathway between adipose tissue and skeletal muscle in the development of insulin resistance, and indicate that adipose tissue-derived miR-27a may play a key role in the development of obesity-triggered insulin resistance in skeletal muscle.
Project description:The clear mechanism of moderate exercise training (Ex) in attenuating muscle loss remains elusive in diabetes. We investigated the effects of moderate exercise training on diabetes-induced nuclear factor-?B (NF-?B) activation and mitochondrial dysfunction. Skeletal muscle size and atrophy signaling pathways were examined in type 2 diabetic db/db mice with or without moderate exercise training (5.2 m/min, 1 h/day, and 5 days/week for a total of 8 weeks). Exercise training decreased serum leptin, MCP-1, and resistin levels in db/db+Ex mice, but it did not reduce symptoms of insulin resistance including hyperglycemia, hyperinsulinemia, and impaired glucose tolerance. Moderate exercise training prevented the loss of muscle mass of tibialis anterior and gastrocnemius muscles in db/db+Ex mice. The average cross-sectional area of tibialis anterior muscle was increased significantly in db/db+Ex mice compared with untrained mice (830.6 vs. 676.5 ?m2). Inhibition of MuRF-1 and K48-linked polyubiquitination was observed in db/db+Ex mice. Exercise training reduced activation of I?B?/NF-?B pathway and lowered IL-6, TNF?, F4/80 (macrophage marker) at mRNA level in db/db+Ex mice compared with untrained mice. Exercise training did not influence FoxO3a phosphorylation and its upstream regulator Akt. Exercise training increased SIRT1 and PGC1? expression and AMPK? and mitochondrial complex IV activities and upregulated genes involved in mitochondrial biogenesis/function including Nrf1, Tfam, and mitochondrial complexes I-V. In conclusion, moderate exercise training inhibits NF?B signaling and activates SIRT1-AMPK?-PGC1? axis, thereby attenuating type 2 diabetes-related muscle atrophy.
Project description:<h4>Aim</h4>des-O-methyllasiodiplodin (DML) from Cerbera manghas has shown antagonistic activity against mineralocorticoid receptor (MR). Considering the involvement of MR in the insulin tolerance, we attempted to investigate the potential of DML in the treatment of type 2 diabetes mellitus (T2DM).<h4>Methods</h4>Surface plasmon resonance (SPR) technology and reporter gene-based assays were used to study protein-small molecule interactions. HepG2 and 3T3-L1 cells were treated with H2O2 (0.2 mmol/L) or aldosterone (10 nmol/L) for 24 h. The expression of MR in the cells was downregulated with siRNA. The anti-inflammatory effect of the compound was evaluated, respectively. db/db mice were administered DML (30 mg·kg(-1)·d(-1)) for 4 weeks. Serum biochemical parameters and insulin sensitivity were examined. The expression levels of pro-inflammatory cytokines (MCP-1, TNF-? and IL-6) and ROS-related genes (NADPH p47 subunit and transcriptional factor PU.1) in adipose tissues and livers were analyzed using real-time RT-PCR.<h4>Results</h4>In HepG2 and 3T3-L1 cells, both H2O2 and aldosterone markedly stimulates the expression of MCP-1, TNF?, IL-6, p47 and PU.1 genes. Co-treatment with DML (10 ?mol/L) significantly reduced the H2O2- or aldosterone-induced expression of these genes. SPR-based assay confirmed the antagonistic activity of DML against the interaction between SRC-1 and MR-LBD. Furthermore, DML decreased aldosterone-induced MR transcriptional activity in a dose-dependent manner. Downregulation of MR with siRNA in the cells prevented or significantly attenuated aldosterone-stimulated expression of these genes, whereas DML did no longer affect the expression of these genes except that of IL-6. Oral administration of DML effectively reduced the levels of blood glucose and glycosylated hemoglobin (HbA1c) in db/db mice. The treatment also rectified the expression of pro-inflammatory factor and ROS-related genes in db/db mice.<h4>Conclusion</h4>DML effectively lowers the blood glucose level in db/db mice possibly via ameliorating the expression of obesity-related pro-inflammatory cytokines, highlighting the potential of the marine natural product as a drug lead for the treatment of metabolic disorders.
Project description:The pathophysiology of type 2 diabetes mellitus (T2D) is characterized by reduced or absent insulin receptor (INSR) responsiveness to its ligand, elevated hepatic glucose output and impaired glucose uptake in peripheral tissues, particularly skeletal muscle. Treatments to reduce hyperglycemia and reestablish normal insulin signaling are much sought after. Any agent which could be orally administered to restore INSR function, in an insulin-independent manner, would have major implications for the management of this global disease. We have discovered a non-peptidyl small molecule, adenosine, 5'-Se-methyl-5'-seleno-, 2',3'-diacetate [referred to as non-peptidyl compound #43 (NPC43)], which restores INSR signaling in the complete absence of insulin. Initial screening of numerous compounds in human HepG2 liver cells revealed that NPC43 significantly inhibited glucose production. The compound was potently anti-hyperglycemic and anti-hyperinsulinemic in vivo, in insulin-resistant T2D Leprdb/db mice, following either acute or chronic treatment by oral gavage and intraperitoneal injection, respectively. The compound acted at the level of INSR and activated it in both liver and skeletal muscle of Leprdb/db mice. In cell culture, the compound activated INSR in both liver and skeletal muscle cells; furthermore, it cooperated with insulin to depress glucose-6-phosphatase catalytic subunit (G6pc) expression and stimulate glucose uptake, respectively. Our results indicated that the compound directly interacted with INSR?, triggering appropriate phosphorylation and activation of the receptor and its downstream targets. Unlike insulin, NPC43 did not activate insulin-like growth factor 1 receptor in either liver or skeletal muscle. We believe this compound represents a potential oral and/or injectable insulin replacement therapy for diabetes and diseases associated with insulin resistance.
Project description:Type 2 diabetes (T2D) patients suffer from dyspnea, which contributes to disease-related morbidity. Although T2D has been reported to induce a catabolic state in skeletal muscle, whether T2D induces muscle wasting in respiratory muscles has not yet been investigated. In this study, we examine the difference in the molecular signaling signature of muscle wasting between the intercostal and gastrocnemius muscles using db/db mice, a well-known diabetic mouse model. Akt phosphorylation was significantly decreased in both the intercostal and gastrocnemius muscles of db/db mice and was accompanied by a decrease in mTORC1 activity. In addition, FoxO phosphorylation was suppressed, and ubiquitin-proteasome degradation, characterized by the level of Atrogin-1 and MuRF1, was subsequently enhanced in both muscle types of db/db mice. An increase in LC3BII levels and a decrease in p62 levels marked the occurrence of substantial autophagy in the gastrocnemius muscle but not in the intercostal muscles of db/db mice. Therefore, we suggest that the signaling events of muscle wasting in the intercostal muscles of db/db mice are different from those in the gastrocnemius muscle of db/db mice.