Docosahexaenoic acid varies in rat skeletal muscle membranes according to fibre type and provision of dietary fish oil.

BACKGROUND: Dietary fish oil provides polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) and is associated with modified oxygen consumption, contractile fatigue and physiological responses to ischaemia or hypoxia in striated muscle. This study systematically investigated the membrane incorporation of fatty acids, with a focus on DHA, into skeletal muscle in relation to functional/metabolic differences and their responsiveness to fish oil doses. METHODS: Male Sprague-Dawley rats were randomised to isoenergetic diets (10% fat by weight). Human Western-style diets were simulated with 5.5% tallow, 2.5% n-6 PUFA sunflower seed oil and 2% olive oil (Control). High-DHA tuna oil exchanged for olive oil provided a Low (0.32%) or moderate (Mod) (1.25%) fish oil diet. Membrane phospholipid fatty acid composition was analysed in samples of five skeletal muscles selected for maximum variation in muscle fibre-type. RESULTS: Concentrations of DHA varied according to muscle fibre type, very strongly associated with fast oxidative glycolytic fibre population (r2 = 0.93; P < 0.01). No relationship was evident between DHA and fast glycolytic or slow oxidative fibre populations. Fish oil diets increased membrane incorporation of DHA in all muscles, mainly at the expense of n-6 PUFA linoleic and arachidonic acid. CONCLUSION: The exquisite responsiveness of all skeletal muscles to as little fish oil as the equivalent of 1-2 fish meals per week in a human diet and the selective relationship to fatigable muscle fibre-types supports an integral role for DHA in muscle physiology, and particularly in fatigue resistance of fast-twitch muscles. SUMMARY: Skeletal muscle fibres vary according to structural, metabolic and neurological characteristics and ultimately influences contractile function. This study sort to determine if the composition of phospholipid polyunsaturated fatty acids (PUFA), incorporated in their membranes, might also differ according to fibre type and when omega-3 PUFA are made available in the diet. We systematically demonstrated that the omega-3 PUFA, docosahexaenoic acid (DHA), incorporated into skeletal muscle membranes well above its provision in the diet and without competitive influence of high omega-6 PUFA concentrations, typical to the Western-style human diet. Notably, incorporation preferentially occurred according to metabolic characteristics of each muscle, supporting the notion that DHA plays an integral role in fast oxidative glycolytic muscle fibres.

Alpha B-crystallin is a major component of glial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of alpha-synuclein filaments. AlphaB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of alphaB-crystallin in GCIs in MSA brains. We also examined the influence of alphaB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed alphaB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that alphaB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for alphaB-crystallin. Proteasome-inhibited cells transfected with GFP-tagged alpha-synuclein resulted in ubiquitin- and alphaB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that alphaB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

The molecular chaperone alpha-crystallin is in kinetic competition with aggregation to stabilize a monomeric molten-globule form of alpha-lactalbumin.

In vivo, alpha-crystallin and other small heat-shock proteins (sHsps) act as molecular chaperones to prevent the precipitation of 'substrate' proteins under stress conditions through the formation of a soluble sHsp-substrate complex. Using a range of different salt conditions, the rate and extent of precipitation of reduced alpha-lactalbumin have been altered. The interaction of alpha-crystallin with reduced alpha-lactalbumin under these various salt conditions was then studied using a range of spectroscopic techniques. Under conditions of low salt, alpha-lactalbumin aggregates but does not precipitate. alpha-Crystallin is able to prevent this aggregation, initially by stabilization of a monomeric molten-globule species of alpha-lactalbumin. It is proposed that this stabilization occurs through weak transient interactions between alpha-crystallin and alpha-lactalbumin. Eventually a stable, soluble high-molecular-mass complex is formed between the two proteins. Thus it appears that a tendency for alpha-lactalbumin to aggregate (but not necessarily precipitate) is the essential requirement for alpha-crystallin-alpha-lactalbumin interaction. In other words, alpha-crystallin interacts with a non-aggregated form of the substrate to prevent aggregation. The rate of precipitation of alpha-lactalbumin is increased significantly in the presence of Na2SO4 compared with NaCl. However, in the former case, alpha-crystallin is unable to prevent this aggregation and precipitation except in the presence of a large excess of alpha-crystallin, i.e. at mass ratios more than 10 times greater than in the presence of NaCl. It is concluded that a kinetic competition exists between aggregation and interaction of unfolding proteins with alpha-crystallin.

The small heat-shock chaperone protein, alpha-crystallin, does not recognize stable molten globule states of cytosolic proteins.

The small heat-shock protein (sHsp), alpha-crystallin, acts as a molecular chaperone by interacting with destabilized 'substrate' proteins to prevent their precipitation from solution under conditions of stress. alpha-Crystallin and all sHsps are intracellular proteins. Similarly to other chaperones, the 'substrate' protein is in an intermediately folded, partly structured molten globule state when it interacts and complexes with alpha-crystallin. In this study, stable molten globule states of the cytosolic proteins, gamma-crystallin and myoglobin, have been prepared. Within the lens, gamma-crystallin naturally interacts with alpha-crystallin and myoglobin and alpha-crystallin are present together in muscle tissue. The molten globule states of gamma-crystallin and myoglobin were prepared by reacting gamma-crystallin with glucose 6-phosphate and by removing the haem group of myoglobin. Following spectroscopic characterisation of these modified proteins, their interaction with alpha-crystallin was examined by a variety of spectroscopic and protein chemical techniques. In both cases, there was no interaction with alpha-crystallin that led to complexation. It is concluded that alpha-crystallin does not recognise stable molten globule states of cytosolic 'substrate' proteins and only interacts with molten globule states of proteins that are on the irreversible pathway towards an aggregated and precipitated form.