Increase in Mitochondrial content after Electrical Pulse Stimulation is dependent on duration of stimulation

Increase in Mitochondrial content after Electrical Pulse Stimulation is dependent on duration of stimulation Daniel Conde B.S.1, Jeffrey D. Covington Ph.D.2, Cecilia Gamboa3 George A. King Ph.D.1, Arild C. Rustan Ph.D.4, Sudip Bajpeyi Ph.D.1. 1 Department of Kinesiology, University of Texas at El Paso, TX; 2Louisiana State University Health Sciences Center, New Orleans, LA; 3Clinical Laboratory Sciences, University of Texas at El Paso; 4Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway. Skeletal Muscle Metabolism Laboratory; Kinesiology; University of Texas at El Paso; El Paso, TX Category: Masters Advisor / Mentor: Bajpeyi, Sudip ([email protected]) ABSTRACT We have previously shown that human skeletal muscle myotubes cultured in vitro, retain in vivo characteristics of the donors. Recent studies indicate that electrical pulse stimulation (EPS) can be used as an exercise mimetic in a cell culture model, and could be beneficial to understand molecular mechanisms underlying exercise training. Purpose: The purpose of this study was to compare acute, moderate and long duration EPS treatments on mitochondrial and lipid content in cultured myotubes. Methods: EPS stimulation was applied to human myotubes cultured from sedentary donors under three conditions: Acute (bipolar pulses of 100 Hz for 200 ms every 5th second; 30V for 60 min) and chronic stimulation (single bipolar pulses of 2 ms; 30V, 1Hz continuously for 24 h or 48 h). Mitochondrial and lipid contents were measured by primary antibody for complex IV and bodipy green dye, respectively, using immunohistochemistry techniques. Fluoroskan ascent microplate reader was used to quantify fluorescence signals. OXPHOS proteins were measured using western immunoblotting. Results: There was no change in lipid or mitochondrial content as assessed by immunohistochemistry after acute EPS stimulation. Chronic stimulation resulted in a significant increase in the mitochondrial content after 24 h (from 0.183 ± 0.02 AU to 0.350 ± 0.03 AU; p=0.008) and 48 h (from 0.290 ± 0.01 AU to 0.337 ± 0.01 AU; p=0.02) of continuous EPS stimulation. OXPHOS proteins increased after 48 h of EPS. There was also a significant increase in lipid content after 48 h of EPS stimulation (from 0.210 ± 0.01 AU to 0.256 ± 0.01 AU; p=0.02). Conclusion: These findings suggest that 48 h of chronic EPS results in an increase in both mitochondrial and lipid contents in human myotubes. The concomitant increase in lipid and mitochondrial content after exercise mimetic EPS stimulation supports the elevated level of intramyocellular lipid and mitochondrial content evident in endurance trained athletes

24 hours of Electrical Pulse Stimulation upregulates GLUT4 and AMPK protein content in human myotubes

Electrical pulse stimulation (EPS), an in vitro exercise mimetic, has been shown to increase mitochondrial and lipid content in cultured human myotubes. We have recently shown that myotubes retain certain in vivo characteristics of the donors. Purpose: We aimed to examine the EPS-induced adaptations in relation to Glucose Transporter Type 4 (GLUT4) and 5’Adenosine Monophosphate-activated Protein Kinase (AMPK) content using human myotubes. Additionally, we examined if the duration of EPS as well as cell harvest times (immediate vs. 24hrs. after the cessation of EPS) plays a role in EPS induced changes in GLUT4 and AMPK content. Methods: EPS was applied to myotubes 24 and 48 hr. (single bipolar pulses of 1 Hz for 2 ms; 30V) and were harvested at two different time points: immediately after (early harvest) and 24hr after (late harvest) the end of stimulation. Total GLUT4 and AMPK content were measured by western immunoblotting. Results: GLUT4 content was ~ 1.7 fold higher after 24 hr. early harvest and ~2.1 fold higher after 48 hr. late harvest stimulation. Total AMPK content was ~3.2 fold higher after 24 hr. early harvest stimulation and ~1.4 fold higher after 48 hr. late harvest stimulation. There was a ~0.6 fold decrease in AMPK after 24 hr. late harvest stimulation. Conclusion: These findings suggest that 24Hr of EPS stimulation upregulates GLUT4 and AMPK protein content. Duration and harvesting time (a reflection of post-exercise recovery) with regards to EPS treatment is a key factor leading to GLUT4 and AMPK content adaptations to exercise in human myotubes

Tree Buffers

In runtime verification, the central problem is to decide if a given program execution violates a given property. In online runtime verification, a monitor observes a program’s execution as it happens. If the program being observed has hard real-time constraints, then the monitor inherits them. In the presence of hard real-time constraints it becomes a challenge to maintain enough information to produce error traces, should a property violation be observed. In this paper we introduce a data structure, called tree buffer, that solves this problem in the context of automata-based monitors: If the monitor itself respects hard real-time constraints, then enriching it by tree buffers makes it possible to provide error traces, which are essential for diagnosing defects. We show that tree buffers are also useful in other application domains. For example, they can be used to implement functionality of capturing groups in regular expressions. We prove optimal asymptotic bounds for our data structure, and validate them using empirical data from two sources: regular expression searching through Wikipedia, and runtime verification of execution traces obtained from the DaCapo test suite

Robustness Testing of Intermediate Verifiers

Program verifiers are not exempt from the bugs that affect nearly every piece of software. In addition, they often exhibit brittle behavior: their performance changes considerably with details of how the input program is expressed-details that should be irrelevant, such as the order of independent declarations. Such a lack of robustness frustrates users who have to spend considerable time figuring out a tool's idiosyncrasies before they can use it effectively. This paper introduces a technique to detect lack of robustness of program verifiers; the technique is lightweight and fully automated, as it is based on testing methods (such as mutation testing and metamorphic testing). The key idea is to generate many simple variants of a program that initially passes verification. All variants are, by construction, equivalent to the original program; thus, any variant that fails verification indicates lack of robustness in the verifier. We implemented our technique in a tool called "mugie", which operates on programs written in the popular Boogie language for verification-used as intermediate representation in numerous program verifiers. Experiments targeting 135 Boogie programs indicate that brittle behavior occurs fairly frequently (16 programs) and is not hard to trigger. Based on these results, the paper discusses the main sources of brittle behavior and suggests means of improving robustness

Altered Skeletal Muscle Lipase Expression and Activity Contribute to Insulin Resistance in Humans

International audienceOBJECTIVE: Insulin resistance is associated with elevated content of skeletal muscle lipids, including triacylglycerols (TAGs) and diacylglycerols (DAGs). DAGs are by-products of lipolysis consecutive to TAG hydrolysis by adipose triglyceride lipase (ATGL) and are subsequently hydrolyzed by hormone-sensitive lipase (HSL). We hypothesized that an imbalance of ATGL relative to HSL (expression or activity) may contribute to DAG accumulation and insulin resistance. RESEARCH DESIGN AND METHODS: We first measured lipase expression in vastus lateralis biopsies of young lean (n = 9), young obese (n = 9), and obese-matched type 2 diabetic (n = 8) subjects. We next investigated in vitro in human primary myotubes the impact of altered lipase expression/activity on lipid content and insulin signaling. RESULTS: Muscle ATGL protein was negatively associated with whole-body insulin sensitivity in our population (r = -0.55, P = 0.005), whereas muscle HSL protein was reduced in obese subjects. We next showed that adenovirus-mediated ATGL overexpression in human primary myotubes induced DAG and ceramide accumulation. ATGL overexpression reduced insulin-stimulated glycogen synthesis (-30%, P < 0.05) and disrupted insulin signaling at Ser1101 of the insulin receptor substrate-1 and downstream Akt activation at Ser473. These defects were fully rescued by nonselective protein kinase C inhibition or concomitant HSL overexpression to restore a proper lipolytic balance. We show that selective HSL inhibition induces DAG accumulation and insulin resistance. CONCLUSIONS: Altogether, the data indicate that altered ATGL and HSL expression in skeletal muscle could promote DAG accumulation and disrupt insulin signaling and action. Targeting skeletal muscle lipases may constitute an interesting strategy to improve insulin sensitivity in obesity and type 2 diabetes

Fatty Acid Incubation of Myotubes From Humans With Type 2 Diabetes Leads to Enhanced Release of β-Oxidation Products Because of Impaired Fatty Acid Oxidation: Effects of Tetradecylthioacetic Acid and Eicosapentaenoic Acid

OBJECTIVE—Increased availability of fatty acids is important for accumulation of intracellular lipids and development of insulin resistance in human myotubes. It is unknown whether different types of fatty acids like eicosapentaenoic acid (EPA) or tetradecylthioacetic acid (TTA) influence these processes