Skeletal Muscle Plasma Membrane Research Articles

Biochemical Toxicological Study of Insulin Overdose in Rats: A Forensic Perspective.

Due to nonspecific pathological changes and the rapid degradation of insulin in postmortem blood samples, the identification of the cause of death during insulin overdose has always been a difficulty in forensic medicine. At present, there is a lack of studies on the toxicological changes and related mechanisms of an insulin overdose, and the specific molecular markers of insulin overdose are still unclear. In this study, an animal model of insulin overdose was established, and 24 SD rats were randomly divided into a control group, insulin overdose group, and a recovery group (n = 8). We detected the biochemical changes and analyzed the toxicological mechanism of an insulin overdose. The results showed that after insulin overdose, the rats developed irregular convulsions, Eclampsia, Opisthotonos, and other symptoms. The levels of glucose, glycogen, and C-peptide in the body decreased significantly, while the levels of lactate, insulin, and glucagon increased significantly. The decrease in plasma K+ was accompanied by the increase in skeletal muscle K+. The PI3K-AKT signaling pathway was significantly activated in skeletal muscle, and the translocation of GLUT4/Na+-K+-ATPase to sarcolemma was significantly increased. Rare glycogenic hepatopathy occurred in the recovery group after insulin overdose. Our study showed that insulin overdose also plays a role in skeletal muscle cells, mainly through the PI3K-Akt signaling pathway. Therefore, the detection of signaling pathway proteins of the skeletal muscle cell membrane GLUT4 and Na+-K+-ATPase has a certain auxiliary diagnostic value for forensic insulin overdose identification. Glycogen detection in the liver and skeletal muscle is important for the diagnosis of insulin overdose, but it still needs to be differentiated from other causes of death. Skeletal muscle has great potential for insulin detection, and the ratio of insulin to the C-peptide (I:C) can determine whether an exogenous insulin overdose is present.

Abstract 059: (Pro)renin Receptor Ablation In Tyrosine Hydroxylase Neurons In The Paraventricular Nucleus Of The Hypothalamus Improves Insulin Sensitivity In Mice Fed A High-fat Diet

We recently reported that neuron-specific (pro)renin receptor (PRR) deletion reduces blood pressure (BP), fasting blood glucose (BG) and improves glucose tolerance in high-fat diet (HFD)-treated mice. To investigate underlying mechanisms, we focused on tyrosine hydroxylase (TH) neurons in the hypothalamic paraventricular nucleus (TH PVN neurons), a largely GABAergic population that expresses PRR and projects to autonomic centers important for BP and BG regulation. The PRR was ablated specifically in the TH-positive neurons in the PVN using Cre-LoxP approach. Radio telemetric BP recording, fasting blood glucose (FBG) and frequently sampled intravenous glucose tolerance test (FSIVGTT) were performed following 6-week HFD (60% calories from fat). HFD induced elevation of BP within the first week ( p < 0.05) and maintained elevated during 6-week protocol; however, there was no difference in BP between TH PVN PRRKO and WT control mice in both sexes following HFD. In male mice, the controls had higher FBG when compared with the TH PVN PRRKO mice (144.1 ± 5.74 vs. 121.9 ± 4.34 mg/dl, p < 0.05) at the end of the 6-week HFD treatment. Interestingly, PRR ablation in the TH PVN neurons did not reduce FBG in female mice. The TH PVN PRRKO mice showed improved glucose handling during FSIVGTT (AUC: 3481 ± 247.5 vs. 4263 ± 153.9, p < 0.05), while there was no difference in insulin secretion (AUC: 1782 ± 339.2 vs. 2324 ± 495.6, p > 0.05) between the two groups suggesting that TH PVN PRR ablation attenuates HFD-induced glucose intolerance through either an increase in insulin sensitivity or an insulin-independent pathway. Detected by western blot, the glucose transporter 4, an insulin-dependent glucose transporter, was higher on the plasma membrane of skeletal muscle in the TH PVN PRRKO mice (1.9 ± 0.2 vs. 1.0 ± 0.1, p < 0.05) compared with the controls. The hepatic mRNA expression of glucose-6-phosphatase, a gluconeogenic gene transcriptional negative-regulated by insulin, was lower in the TH PVN PRRKO (0.60 ± 0.07 vs. 0.95 ± 0.10, p < 0.05) compared with the control mice, suggesting improvement of insulin sensitivity in the TH PVN PRRKO mice. In conclusion, PRR ablation in TH PVN neurons attenuates FBG and glucose tolerance through improvement of insulin sensitivity without impacting BP.

An early, reversible cholesterolgenic etiology of diet-induced insulin resistance

ObjectiveA buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance. MethodsC57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet. ResultsSaline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM. ConclusionsThese data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development.

In Vivo and In Vitro Antidiabetic Efficacy of Aqueous and Methanolic Extracts of Orthosiphon Stamineus Benth

Orthosiphon stamineus is a popular folk herb used to treat diabetes and some other disorders. Previous studies have shown that O. stamineus extracts were able to balance blood glucose levels in diabetic rat animal models. However, the antidiabetic mechanism of O. stamineus is not fully known. This study was carried out to test the chemical composition, cytotoxicity, and antidiabetic activity of O. stamineus (aerial) methanol and water extracts. GC/MS phytochemical analysis of O. stamineus methanol and water extracts revealed 52 and 41 compounds, respectively. Ten active compounds are strong antidiabetic candidates. Oral treatment of diabetic mice with O. stamineus extracts for 3 weeks resulted significant reductions in blood glucose levels from 359 ± 7 mg/dL in diabetic non-treated mice to 164 ± 2 mg/dL and 174 ± 3 mg/dL in water- and methanol-based-extract-treated mice, respectively. The efficacy of O. stamineus extracts in augmenting glucose transporter-4 (GLUT4) translocation to the plasma membrane (PM) was tested in a rat muscle cell line stably expressing myc-tagged GLUT4 (L6-GLUT4myc) using enzyme-linked immunosorbent assay. The methanol extract was more efficient in enhancing GLUT4 translocation to the PM. It increased GLUT4 translocation at 250 µg/mL to 279 ± 15% and 351 ± 20% in the absence and presence of insulin, respectively. The same concentration of water extract enhanced GLUT4 translocation to 142 ± 2.5% and 165 ± 5% in the absence and presence of insulin, respectively. The methanol and water extracts were safe up to 250 µg/mL as measured with a Methylthiazol Tetrazolium (MTT) cytotoxic assay. The extracts exhibited antioxidant activity as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. O. stamineus methanol extract reached the maximal inhibition of 77 ± 10% at 500 µg/mL, and O. stamineus water extract led to 59 ± 3% inhibition at the same concentration. These findings indicate that O. stamineus possesses antidiabetic activity in part by scavenging the oxidants and enhancing GLUT4 translocation to the PM in skeletal muscle.

Inhibitory machinery for the functional dystroglycan glycosylation.

Dystroglycan (DG), a muscular transmembrane protein, plays a critical role in transducing extracellular matrix-derived signals to the cytoskeleton and provides physical strength to skeletal muscle cell membranes. The extracellular domain of DG, α-DG, displays unique glycosylation patterns. Fully functional glycosylation is required for this domain to interact with components of extracellular matrices, including laminin. One of the unique sugar compositions found in such functional glycans on DG is two ribitol phosphates that are transferred by the sequential actions of fukutin (FKTN) and fukutin-related protein (FKRP), which use CDP-ribitol as a donor substrate. These are then further primed for matriglycan biosynthesis. A recent in vitro study reported that glycerol phosphate could be similarly added to α-DG by FKTN and FKRP if they used CDP-glycerol (CDP-Gro) as a donor substrate. However, the physiological relevance of these findings remains elusive. Imae et al. addressed the knowledge gap regarding whether CDP-Gro is present in mammals and how CDP-Gro is synthesized and functions in mammals.

Reactive Oxygen and Nitrogen Species (RONS) and Cytokines—Myokines Involved in Glucose Uptake and Insulin Resistance in Skeletal Muscle

Insulin resistance onset in skeletal muscle is characterized by the impairment of insulin signaling, which reduces the internalization of glucose, known as glucose uptake, into the cell. Therefore, there is a deficit of intracellular glucose, which is the main source for energy production in the cell. This may compromise cellular viability and functions, leading to pathological dysfunction. Skeletal muscle fibers continuously generate reactive oxygen and nitrogen species (RONS). An excess of RONS produces oxidative distress, which may evoke cellular damage and dysfunction. However, a moderate level of RONS, which is called oxidative eustress, is critical to maintain, modulate and regulate cellular functions through reversible interactions between RONS and the components of cellular signaling pathways that control those functions, such as the facilitation of glucose uptake. The skeletal muscle releases peptides called myokines that may have endocrine and paracrine effects. Some myokines bind to specific receptors in skeletal muscle fibers and might interact with cellular signaling pathways, such as PI3K/Akt and AMPK, and facilitate glucose uptake. In addition, there are cytokines, which are peptides produced by non-skeletal muscle cells, that bind to receptors at the plasma membrane of skeletal muscle cells and interact with the cellular signaling pathways, facilitating glucose uptake. RONS, myokines and cytokines might be acting on the same signaling pathways that facilitate glucose uptake in skeletal muscle. However, the experimental studies are limited and scarce. The aim of this review is to highlight the current knowledge regarding the role of RONS, myokines and cytokines as potential signals that facilitate glucose uptake in skeletal muscle. In addition, we encourage researchers in the field to lead and undertake investigations to uncover the fundamentals of glucose uptake evoked by RONS, myokines, and cytokines.

GSK3α phosphorylates dynamin-2 to promote GLUT4 endocytosis in muscle cells.

Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to plasma membrane of skeletal muscle is critical for postprandial glucose uptake; however, whether the internalization of GLUT4 is also regulated by insulin signaling remains unclear. Here, we discover that the activity of dynamin-2 (Dyn2) in catalyzing GLUT4 endocytosis is negatively regulated by insulin signaling in muscle cells. Mechanistically, the fission activity of Dyn2 is inhibited by binding with the SH3 domain of Bin1. In the absence of insulin, GSK3α phosphorylates Dyn2 to relieve the inhibition of Bin1 and promotes endocytosis. Conversely, insulin signaling inactivates GSK3α and leads to attenuated GLUT4 internalization. Furthermore, the isoform-specific pharmacological inhibition of GSK3α significantly improves insulin sensitivity and glucose tolerance in diet-induced insulin-resistant mice. Together, we identify a new role of GSK3α in insulin-stimulated glucose disposal by regulating Dyn2-mediated GLUT4 endocytosis in muscle cells. These results highlight the isoform-specific function of GSK3α on membrane trafficking and its potential as a therapeutic target for metabolic disorders.

Insulin, Muscle Glucose Uptake, and Hexokinase: Revisiting the Road Not Taken.

Research conducted over the last 50 yr has provided insight into the mechanisms by which insulin stimulates glucose transport across the skeletal muscle cell membrane Transport alone, however, does not result in net glucose uptake as free glucose equilibrates across the cell membrane and is not metabolized. Glucose uptake requires that glucose is phosphorylated by hexokinases. Phosphorylated glucose cannot leave the cell and is the substrate for metabolism. It is indisputable that glucose phosphorylation is essential for glucose uptake. Major advances have been made in defining the regulation of the insulin-stimulated glucose transporter (GLUT4) in skeletal muscle. By contrast, the insulin-regulated hexokinase (hexokinase II) parallels Robert Frost's "The Road Not Taken." Here the case is made that an understanding of glucose phosphorylation by hexokinase II is necessary to define the regulation of skeletal muscle glucose uptake in health and insulin resistance. Results of studies from different physiological disciplines that have elegantly described how hexokinase II can be regulated are summarized to provide a framework for potential application to skeletal muscle. Mechanisms by which hexokinase II is regulated in skeletal muscle await rigorous examination.

An integrative proteomics metabolomics based strategy reveals the mechanisms involved in wasp sting induced acute kidney injury

The pathophysiological mechanisms involved in wasp-sting-induced acute kidney injury (AKI) remain largely unknown. Here, we combined proteomics and metabolomics to investigate the mechanisms behind multiple wasp sting-induced AKI. Interestingly, we found many differentially abundant proteins in the serum of AKI group compared with that of the non-AKI and control groups, involved in several metabolic pathways and the regulation of cellular processes. In addition, we also detected differentially abundant metabolites in the AKI group; among them many were involved in the glycerophospholipid metabolic pathway (the key pathway in the context of AKI): 50 metabolites, all downregulated in the AKI group. Importantly, the convergent analysis of metabolomics and proteomics data revealed that biomarkers of rhabdomyolysis (CA 3, MYL3, and LDH) and hemolysis (ALT and LDH) were integrated into a regulatory network with phospholipid metabolism products in the AKI group, indicating that wasp sting-induced AKI is secondary to rhabdomyolysis and intravascular hemolysis. Of note, such a phenotype suggests the disruption of the membrane of skeletal muscle cells and red blood cells mediated by the phospholipase A1 (PLA1), PLA2, and mastoparan in the wasp venom, via the disruption of membrane glycerophospholipids. Overall, our results highlight a potential new mechanism behind wasp sting-induced AKI and suggest that PLA inhibitors may be potential agents for the treatment of this condition.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

Rho Family GTPases and Rho GEFs in Glucose Homeostasis.

Dysregulation of glucose homeostasis leading to metabolic syndrome and type 2 diabetes is the cause of an increasing world health crisis. New intriguing roles have emerged for Rho family GTPases and their Rho guanine nucleotide exchange factor (GEF) activators in the regulation of glucose homeostasis. This review summates the current knowledge, focusing in particular on the roles of Rho GEFs in the processes of glucose-stimulated insulin secretion by pancreatic β cells and insulin-stimulated glucose uptake into skeletal muscle and adipose tissues. We discuss the ten Rho GEFs that are known so far to regulate glucose homeostasis, nine of which are in mammals, and one is in yeast. Among the mammalian Rho GEFs, P-Rex1, Vav2, Vav3, Tiam1, Kalirin and Plekhg4 were shown to mediate the insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane and/or insulin-stimulated glucose uptake in skeletal muscle or adipose tissue. The Rho GEFs P-Rex1, Vav2, Tiam1 and β-PIX were found to control the glucose-stimulated release of insulin by pancreatic β cells. In vivo studies demonstrated the involvement of the Rho GEFs P-Rex2, Vav2, Vav3 and PDZ-RhoGEF in glucose tolerance and/or insulin sensitivity, with deletion of these GEFs either contributing to the development of metabolic syndrome or protecting from it. This research is in its infancy. Considering that over 80 Rho GEFs exist, it is likely that future research will identify more roles for Rho GEFs in glucose homeostasis.

Relationship between the Sports Ability of Sports Tennis Players and Minerals and Vitamins

High-intensity training and competition consume a lot of physical energy for tennis players. It is not enough to rely on exercise means, physics means and sleep for recovery. The substances consumed during exercise are mainly supplemented by the intake of nutrients in the diet. Although energy intake basically meets the demand, there are situations where carbohydrate intake is too low and protein intake is too high. In the intake of minerals and vitamins, half of athletes do not meet the recommended intake standards. Therefore, this article uses minerals and vitamins as the starting point to explore their impact on sports tennis athletes’ athletic ability, and conduct simulation experiments on our city’s tennis athletes. The experimental results show that only 54.3% of players know the correct three-meal ratio. The high concentration of vitamin C reduces the lipid peroxidation damage of the skeletal muscle cell membrane, thereby reducing the degree of muscle soreness and speeding up recovery. High-intensity training can increase the excretion of Ca in urine and decrease of Ca in blood below the clinical standard. Supplementing Mg additives can effectively enhance athletes' skeletal muscle and cardiac muscle function. Sports tennis players can enhance immune function and relieve fatigue and muscle aches by supplementing vitamin C.

Sepsis impairs aggregation of nicotinic acetylcholine receptors on murine skeletal muscle cell membranes by inhibiting AKT/GSK3β phosphorylation

To investigate the role of the protein-serine-threonine kinase (AKT)/glycogen synthase kinase 3β (GSK3β) signaling pathway in nicotinic acetylcholine receptors (nAChRs) aggregation disorder on skeletal muscle cell membranes induced by sepsis. Mouse C2C12 myoblasts were differentiated into myotubes by horse serum, and then C2C12 myotubes were randomly divided into four groups: the Sham group treated with serum from sham-operated mice, the Sepsis group treated with serum from septic mice, the Sepsis+D group treated with serum from septic mice and dimethyl sulfoxide (DMSO), the Sepsis+SB group treated with serum from septic mice and GSK3β inhibitor SB216763. Agrin was added into the cell culture to induce nAChRs aggregation before the treatment. After serum treatment for 5.5 h, the myotubes were examined for nAChRs clusters using Alexa Fluor 594-conjugated α-bungarotoxin (α- BTX). The expression levels of AKT, GSK3β and CLIP- associated protein 2 (CLASP2) and the phosphorylation of AKT, GSK3β were examined with Western blotting. The phosphorylation of CLASP2 and the interaction between CLASP2 and α-tubulin were detected with co-immunoprecipitation (Co-IP) assay. Compared with the serum from sham-operated mice, the serum from septic mice caused significant reduction in the area and density of nAChRs clusters on C2C12 myotubes, lowered the levels of phosphorylated AKT (p-AKT) and phosphorylated GSK3β (p-GSK3β), increased the expression of phosphorylated CLASP2 (p-CLASP2), and obviously reduced the binding between CLASP2 and α-tubulin. Compared with DMSO, SB216763 significantly increased the area and density of nAChRs clusters on C2C12 myotubes treated with serum from septic mice, decreased the expression of p-CLASP2, and enhanced the interaction between CLASP2 and α-tubulin. Septic mouse serum impairs nAChRs aggregation on C2C12 myotubes possibly by suppressing AKT/GSK3β phosphorylation to cause reduced interaction between CLASP2 and α-tubulin.

The effect of electric field, magnetic field, and infrared ray combination to reduce HOMA-IR index and GLUT 4 in diabetic model of Mus musculus

Diabetes mellitus (DM) is a metabolic disorder characterized by high blood glucose level (hyperglycemia). Type 2 diabetes mellitus is mainly featured by low cell sensitivity towards insulin stimulation, caused by ectopic fat storage. Insulin resistance can be quantified from high number of HOMA-IR index and observed from glucose transporter 4 (GLUT-4) translocation on membrane of skeletal muscle cells. Combined treatment of electric field, magnetic field, and infrared ray have potential to reduce insulin resistance due to improving blood circulation and increasing intracellular Ca2+ level. The aim of study was to determine the effect of electric field, magnetic field, and infrared ray combination to lower insulin resistance in the type II diabetic model of Mus musculus. This study used 30 adult male mice strain BALB/c. Diabetes was induced using high-fat diet/streptozotocin method until random blood glucose level reached > 200 mg/dL. Diabetic mice were then exposed to electrical field (static and dynamic), magnetic field (static and induce), and infrared ray (with or without infrared ray) combination therapy 15 min daily for 28 days. Fasting blood glucose level, plasma insulin level, HOMA-IR index, and membrane GLUT-4 density after treatment were analyzed statistically at α = 0.05. Result showed that exposure combination of electrical field, magnetic field, and infrared were found to be able to lower fasting blood glucose level and HOMA-IR index significantly, but plasma insulin level and GLUT-4 density were not found to be significantly different compared to diabetic control. Based on current study result, the best combination for reducing insulin resistance in diabetic mice is BsEsI (combination of static magnetic field (Bs), static electric field (Es), with infrared (I)), indicated by lowest HOMA-IR compared to other groups. Exposure to combination of magnetic field, electrical field, and infrared resulted in lowering fasting blood glucose level and HOMA-IR index in diabetic mice, indicating reduced insulin resistance.

Overactivation of the sodium-calcium exchanger and transient receptor potential in anesthesia-induced malignant hyperthermia.

Malignant hyperthermia is a pharmacogenetic disorder, which is an uncommon but frequently fatal intricacy of inhalation anesthesia in man. It causes a quick rise in body temperature to highly irreversible levels, which causes death in around three of four cases. The trigger anesthetics cause an anomalous, continued ascent in myoplasmic calcium levels. Possible mechanisms by which continuous release of sodium, calcium from skeletal muscle plasma membrane and sarcoplasmic reticulum stores respectively can produce the profound hyperthermia are discussed.

Hydrogen Sulfide Improves Aberrant Gastric Smooth Muscle Function in Duchenne Muscular Dystrophy Mice

Deficiency of dystrophin, a cytoskeletal protein localized in the inner face of the plasma membrane in skeletal, cardiac and smooth muscle, results in Duchenne Muscular Dystrophy (DMD). Through its...

Hydrogen Sulfide Improves Aberrant Gastric Smooth Muscle Function in Duchenne Muscular Dystrophy Mice

Deficiency of dystrophin, a cytoskeletal protein localized in the inner face of the plasma membrane in skeletal, cardiac and smooth muscle, results in Duchenne Muscular Dystrophy (DMD). Through its interactions with extracellular matrix and plasma membrane proteins, dystrophin plays a role in contraction and signal transduction. In DMD, gastrointestinal disorders such as gastric dilation and intestinal pseudo‐obstruction resulting from altered motility have been reported. The role of dystrophin in the regulation of contractile protein expression and smooth muscle function, per se, is not known. Studies have suggested that inflammation contributes to the pathophysiology of DMD. Exogenous H2S had been shown to exert beneficial cardiovascular and gastrointestinal functions, probably via exerting anti‐inflammatory actions.AimTo test the hypothesis that a lack of dystrophin causes a decrease in contractile protein expression and smooth muscle function and that treatment with H2S restores the effects of dystrophin deficiency.MethodsThe role of dystrophin was examined using mice deficient in dystrophin alone (mdx) and mice deficient in dystrophin plus telomerase RNA (mdx/mTR), which exhibit increased disease severity. The effect of an orally‐active, slow releasing H2S agent (SG1002) was tested in mdx/mTR mice (40 mg/kg body weight in chow/every 3 days starting from 3 weeks to 9 months). Contraction in response to acetylcholine (ACh) was measured in gastric muscle strips isolated from mdx, mdx/mTR and SG1002‐treated mdx/mTR mice. Age‐matched control mice were used for each group. Contraction was also measured in muscle cells isolated from the stomach of control and mdx mice by scanning micrometry and expressed as the percent decrease in muscle cell length. Expression of contractile proteins such as smoothelin, caldesmon, calponin and tropomyosin was measured by qRT‐PCR and western blot.ResultsAcetylcholine‐induced contraction was reduced in muscle strips from mdx/mTR mice (19 ± 5 mN/100 mg tissue) compared to age‐matched 9‐month old control mice (34 ± 7 mN/100 mg tissue). Treatment of mdx/mTR mice with SG1002 restored contraction to above normal levels (56 ± 8 mN/100 mg tissue). Contraction was also reduced in mdx mice (22 ± 8 mN/100 mg tissue) compared to age‐matched 3‐month old control mice (40 ± 5 mN/100 mg tissue). Acetylcholine‐induced contraction was also decreased in gastric muscle cells from mdx mice (25 ± 3% decrease in cell length) compared to control mice (41 ± 5% decrease in cell length). Expression of contractile proteins was decreased in gastric smooth muscle from mdx and mdx/mTR mice compared to age‐matched controls. Treatment of mdx/mTR mice with SG1002 restored expression of contractile proteins to levels similar to control.ConclusionThe results support our hypothesis that dystrophin deficiency affects contractile protein expression leading to decreased muscle contraction. Furthermore, treatment with H2S restores gastric smooth muscle function and contractile protein expression suggesting therapeutic potential of H2S in the treatment of motility disorders in DMD.Support or Funding InformationSupported by DK15564, DK28300, DK34153, and HL133167.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Forensic Postmortem Interval Estimation from Skeletal Muscle Tissue: A Lipidomics Approach

Lipidomic analyses of human skeletal muscle tissue were conducted to detect biomarkers of time-dependent postmortem degradation and to test the predictive capacity of lipids in human skeletal muscle cell membranes. High-resolution mass spectrometry analytical platforms were used to isolate phospholipids in muscle cell membranes that are specific to the corpse tissues and not invading microbes, thus eliminating potential noise from the surrounding microenvironment. The most consistently extracted cell membrane phospholipids were phosphatidylglycerol (PG) 34:0 and phosphatidylethanolamine (PtdE) 36:4. The actual accumulated degree days of all validation samples fell within the 95% prediction interval limits for the simple linear regression models with PtdE 36:4 and PG 34:0, though the prediction intervals for the latter were wider. The analysis requires only a small amount of tissue, is less subjective than visual methods for estimating postmortem interval, and is robust to drastic fluctuations in temperature. Sophisticated quantitative methods for estimating PMI from biomolecules unique to the corpse, and the human microbiome may provide a means of overcoming the geographic limitations of methods based on subjective visual observations of decomposition changes.

LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation

Adaption of skeletal muscle to endurance exercise includes PPARδ- and AMP-activated protein kinase (AMPK)/PPARγ coactivator 1α-mediated transcriptional responses that result in increased oxidative capacity and conversion of glycolytic to more oxidative fiber types. These changes are associated with whole-body metabolic alterations including improved glucose handling and resistance to obesity. Increased DHA (22:6n-3) content in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is also reported in endurance exercise-trained glycolytic muscle; however, the DHA-metabolizing enzymes involved and the biological significance of the enhanced DHA content are unknown. In the present study, we identified lysophosphatidic acid acyltransferase (LPAAT)3 as an enzyme that was upregulated in myoblasts during in vitro differentiation and selectively incorporated DHA into PC and PE. LPAAT3 expression was increased by pharmacological activators of PPARδ or AMPK, and combination treatment led to further increased LPAAT3 expression and enhanced incorporation of DHA into PC and PE. Our results indicate that LPAAT3 was upregulated by exercise-induced signaling pathways and suggest that LPAAT3 may also contribute to the enhanced phospholipid-DHA content of endurance-trained muscles. Identification of DHA-metabolizing enzymes in the skeletal muscle will help to elucidate broad metabolic effects of DHA.

Blackcurrant Extract Ameliorates Hyperglycemia in Type 2 Diabetic Mice in Association with Increased Basal Secretion of Glucagon-Like Peptide-1 and Activation of AMP-Activated Protein Kinase.

Blackcurrants are berries that contain high levels of anthocyanins, particularly delphinidin 3-rutinoside (D3R). Several studies have reported that the consumption of blackcurrant extract (BCE) lowers blood glucose levels and ameliorates glucose tolerance, but the mechanism underlying this effect remains unclear. Glucagon-like peptide-1 (GLP-1) and AMP-activated protein kinase (AMPK) are considered one of the most significant molecular targets for the prevention and treatment of type 2 diabetes. In this study, we showed that dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in type 2 diabetic mice (KK-Ay). The basal GLP-1 concentration in plasma was significantly increased in the BCE group accompanied by upregulation of prohormone convertase 1/3 (PC1/3), the enzyme that processes intestinal proglucagon. Moreover, the level of phospho-AMPKα protein in skeletal muscle was significantly increased in the BCE group, and this was increase accompanied by significant upregulation of glucose transporter 4 (Glut4) proteins in the plasma membrane of BCE group. In conclusion, dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in association with increased basal GLP-1 concentration in plasma, upregulation of PC1/3 expression, and translocation of Glut4 to the plasma membrane of skeletal muscle in type 2 diabetic mice; furthermore, these effects were accompanied by activation of AMPK. Our findings demonstrated that D3R-rich BCE may help prevent diabetes and allow the dosages of diabetes drugs to be reduced.