An investigation into the P13-K/AKT signalling pathway in TNF-a-induced muscle proeolysis in L6 myotubes
Date
2008-12
Authors
Sishi, Balindiwe J. N.
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized by
a reduction in muscle fibre cross-sectional area as well as protein content, reduced force,
elevated fatigability and insulin resistance. It seems to be a highly ordered and regulated
process and signs of this condition are often seen in inflammatory conditions such as cancer,
AIDS, diabetes and chronic heart failure (CHF). It has long been understood that an
imbalance between protein degradation (increase) and protein synthesis (decrease) both
contribute to the overall loss of muscle protein. Although the triggers that cause atrophy are
different, the loss of muscle mass in each case involves a common phenomenon that induces
muscle proteolysis. It is becoming evident that interactions among known proteolytic systems
(ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factors
such as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases in
which skeletal muscle proteolysis presents a lethal threat. There is an increasing body of
evidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number of
clinical settings but the mechanisms mediating its effects are not completely understood. It is
also now apparent that the transcription factor NF-κB is a key intracellular signal transducer
in muscle catabolic conditions. This study investigated the various proposed signalling
pathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, in
order to synthesize our current understanding of the molecular regulation of muscle atrophy.
Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standard
conditions where after reaching ± 60-65% confluency levels, differentiation was induced for a
maximum of 8 days. During the last 2 days, myotubes were incubated with increasing
concentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24
and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTT
assay and Trypan Blue exclusion technique. Morphological assessment of cell death was
conducted using the Hoechst 33342 and Propidium Iodide staining method. Detection of
apoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used for
the measurement of cell size. In order to determine the source and amount of ROS production,
MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed by
immunohistochemistry. PI3-K activity was calculated by using an ELISA assay and the
expression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate the
quantity of ROS production in TNF-α-induced muscle atrophy.
Results and Discussion: Incubation of L6 myotubes with increasing concentrations of
recombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic to
the cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis and
necrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF-
α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidly
activated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation of
this pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1
and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-α
concentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well as
significant reductions in differentiation markers. Western blot analysis also showed that
certain MAPKs are activated in response to TNF-α. No profound changes were observed with
ROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROS
production. These findings suggest that TNF-α may elicit strong catabolic effects on L6
myotubes in a dose and time dependent manner.
Conclusion: These observations suggest that TNF-α might have beneficial effects in
skeletal muscle in certain circumstances. This beneficial effect however is limited by several
aspects which include the concentration of TNF-α, cell type, time of exposure, culture
conditions, state of the cell (disturbed or normal) and the cells stage of differentiation. The
effect of TNF-α can be positive or negative depending on the concentration and time points
analysed. This action is mediated by various signal transduction pathways that are thought to
cooperate with each other. More understanding of these pathways as well as their subsequent
upstream and downstream constituents is obligatory to clarify the central mechanism/s that
control physiological and pathophysiological effects of TNF-α in skeletal muscle.
Description
Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008.
Keywords
Skeletal muscle atrophy, Muscle proteolysis, Musculoskeletal system -- Diseases, Dissertations -- Physiology (Human and animal), Theses -- Physiology (Human and animal), Muscular atrophy, Tumor necrosis factor