Both full length-cholesteryl ester transfer protein and exon 9-deleted cholesteryl ester transfer protein promote triacylglycerol storage in cultured hepatocytes.

We previously reported that overexpression of full-length cholesteryl ester transfer protein (FL-CETP), but not its exon 9-deleted variant (∆E9-CETP), in an adipose cell line reduces their triacylglycerol (TAG) content. This provided mechanistic insight into several in vivo studies where FL-CETP levels are inversely correlated with adiposity. However, increased FL-CETP is also associated with elevated hepatic lipids, suggesting that the effect of CETP on cellular lipid metabolism may be tissue-specific. Here, we directly investigated the role of FL-CETP and ∆E9-CETP in hepatic lipid metabolism. FL- or ∆E9-CETP was overexpressed in HepG2-C3A by adenovirus transduction. Overexpression of either FL or ∆E9-CETP in hepatocytes increased cellular TAG mass by 25% but reduced TAG secretion. This cellular TAG was contained in larger and more numerous lipid droplets. Analysis of TAG synthetic and catabolic pathways showed that this elevated TAG content was due to increased incorporation of fatty acid into TAG (24%), and higher de novo synthesis of fatty acid (50%) and TAG from acetate (40%). siRNA knockdown of CETP had the opposite effect on TAG synthesis and lipogenesis, and decreased cellular TAG. This novel increase in cellular TAG by FL-CETP overexpression was reproduced in Caco-2 intestinal epithelial cells. We conclude that, unlike that seen in adipocyte cells, overexpression of either CETP isoform in lipoprotein-secreting cells promotes the accumulation of TAG. These data suggest that the in vivo correlation between CETP levels and hepatic steatosis can be explained, in part, by a direct effect of CETP on hepatocyte cellular metabolism.

Essential role of DNA-PKcs and plasminogen for the development of doxorubicin-induced glomerular injury in mice.

Susceptibility to doxorubicin-induced nephropathy (DIN), a toxic model for the induction of proteinuria in mice, is related to the single-nucleotide polymorphism (SNP) C6418T of the Prkdc gene encoding for the DNA-repair enzyme DNA-PKcs. In addition, plasminogen (Plg) has been reported to play a role in glomerular damage. Here, we investigated the interdependence of both factors for the development of DIN. Genotyping confirmed the SNP of the Prkdc gene in C57BL/6 (PrkdcC6418/C6418) and 129S1/SvImJ (PrkdcT6418/T6418) mice. Intercross of heterozygous 129SB6F1 mice led to 129SB6F2 hybrids with Mendelian inheritance of the SNP. After doxorubicin injection, only homozygous F2 mice with PrkdcT6418/T6418 developed proteinuria. Genetic deficiency of Plg (Plg-/-) in otherwise susceptible 129S1/SvImJ mice led to resistance to DIN. Immunohistochemistry revealed glomerular binding of Plg in Plg+/+ mice after doxorubicin injection involving histone H2B as Plg receptor. In doxorubicin-resistant C57BL/6 mice, Plg binding was absent. In conclusion, susceptibility to DIN in 129S1/SvImJ mice is determined by a hierarchical two-hit process requiring the C6418T SNP in the Prkdc gene and subsequent glomerular binding of Plg. This article has an associated First Person interview with the first author of the paper.

Plasminogen-induced foam cell formation by macrophages occurs through a histone 2B (H2B)-PAR1 pathway and requires integrity of clathrin-coated pits.

OBJECTIVE: Plasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease-activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen-mediated FCF by macrophages and if PARs are involved in this process. APPROACH AND RESULTS: Treating macrophages with plasminogen increases their oxidized low-density lipoprotein uptake and plasminogen-mediated foam cell formation (Plg-FCF) significantly. The magnitude of Plg-FCF correlates with cell-surface expression of the H2B level. H2B blockade or downregulation reduces Plg-FCF, whereas its overexpression or high endogenous levels increases Plg-FCF. Modulating PAR1 level in mouse macrophages affects Plg-FCF. Activation/overexpression of PAR1 increases and its blockade/knockdown reduces this response. Confocal imaging indicates that both H2B and PAR1 colocalize with clathrin coated pits on the surface of macrophages, and reducing expression of clathrin or interfering with the clathrin-coated pits integrity reduces Plg-FCF. CONCLUSION: Our data indicate that the magnitude of Plg-FCF by macrophages is proportional to the H2B levels and demonstrate for the first time that PAR1 is involved in this process and that the integrity of clathrin-coated pits is required for the full effect of Plg-induced FCF.

Exon 9-deleted CETP inhibits full length-CETP synthesis and promotes cellular triglyceride storage.

Cholesteryl ester transfer protein (CETP) exists as full-length (FL) and exon 9 (E9)-deleted isoforms. The function of E9-deleted CETP is poorly understood. Here, we investigated the role of E9-deleted CETP in regulating the secretion of FL-CETP by cells and explored its possible role in intracellular lipid metabolism. CETP overexpression in cells that naturally express CETP confirmed that E9-deleted CETP is not secreted, and showed that cellular FL- and E9-deleted CETP form an isolatable complex. Coexpression of CETP isoforms lowered cellular levels of both proteins and impaired FL-CETP secretion. These effects were due to reduced synthesis of both isoforms; however, the predominate consequence of FL- and E9-deleted CETP coexpression is impaired FL-CETP synthesis. We reported previously that reducing both CETP isoforms or overexpressing FL-CETP impairs cellular triglyceride (TG) storage. To investigate this further, E9-deleted CETP was expressed in SW872 cells that naturally synthesize CETP and in mouse 3T3-L1 cells that do not. E9-deleted CETP overexpression stimulated SW872 triglyceride synthesis and increased stored TG 2-fold. Expression of E9-deleted CETP in mouse 3T3-L1 cells produced a similar lipid phenotype. In vitro, FL-CETP promotes the transfer of TG from ER-enriched membranes to lipid droplets. E9-deleted CETP also promoted this transfer, although less effectively, and it inhibited the transfer driven by FL-CETP. We conclude that FL- and E9-deleted CETP isoforms interact to mutually decrease their intracellular levels and impair FL-CETP secretion by reducing CETP biosynthesis. E9-deleted CETP, like FL-CETP, alters cellular TG metabolism and storage but in a contrary manner.

Identification of a hormone response element that mediates suppression of APOF by LXR and PPARα agonists.

Apolipoprotein F (ApoF) regulates cholesteryl ester transfer protein activity. We previously observed that hepatic APOF mRNA levels are decreased by high fat, cholesterol-enriched diets. Here we show in human liver C3A cells that APOF mRNA levels are reduced by agonists of LXR and PPARα nuclear receptors. This negative regulation requires co-incubation with the RXR agonist, retinoic acid. Bioinformatic analysis of the ~2 kb sequence upstream of the APOF promoter identified one potential LXR and 4 potential PPARα binding sites clustered between nucleotides -2007 and -1961. ChIP analysis confirmed agonist-dependent binding of LXRα, PPARα, and RXRα to this hormone response element complex (HREc). A luciferase reporter containing the 2 kb 5' APOF sequence was negatively regulated by LXR and PPARα ligands as seen in cells. This regulation was maintained in constructs lacking the ~1700 nucleotides between the HREc and the APOF proximal promoter. Mutations of the HREc that disrupted LXRα and PPARα binding led to the loss of reporter construct inhibition by agonists of these nuclear receptors. siRNA knockdown studies showed that APOF gene regulation by LXRα or PPARα agonists did not require an interaction between these two nuclear receptors. Thus, APOF is subject to negative regulation by agonist-activated LXR or PPARα nuclear receptors binding to a regulatory element ~1900 bases 5' to the APOF promoter. High fat, cholesterol-enriched diets likely reduce APOF gene expression via these receptors interacting at this regulatory site.

ApoF knockdown increases cholesteryl ester transfer to LDL and impairs cholesterol clearance in fat-fed hamsters.

Cholesteryl ester transfer protein (CETP) regulates intravascular lipoprotein metabolism. In vitro studies indicate that ApoF alters CETP function by inhibiting its activity with LDL. To explore in vivo the complexities driving ApoF's effects on CETP, we developed a siRNA-based hamster model of ApoF knockdown. In both male and female hamsters on chow- or fat-fed diets, we measured lipoprotein levels and composition, determined CETP-mediated transfer of cholesteryl esters (CEs) between lipoproteins, and quantified reverse cholesterol transport (RCT). We found that apoF knockdown in chow-fed hamsters had no effect on lipoprotein levels or composition, but these ApoF-deficient lipoproteins supported 50-100% higher LDL CETP activity in vitro. ApoF knockdown in fat-fed male hamsters created a phenotype in which endogenous CETP-mediated CE transfer from HDL to LDL increased up to 2-fold, LDL cholesterol increased 40%, HDL declined 25%, LDL and HDL lipid compositions were altered, and hepatic LDLR gene expression was decreased. Diet-induced hypercholesterolemia obscured this phenotype on occasion. In fat-fed female hamsters, ApoF knockdown caused similar but smaller changes in plasma CETP activity and LDL cholesterol. Notably, ApoF knockdown impaired HDL RCT in fat-fed hamsters but increased sterol excretion in chow-fed animals. These in vivo data validate in vitro findings that ApoF regulates lipid transfer to LDL. The consequences of ApoF knockdown on lipoproteins and sterol excretion depend on the underlying lipid status. By minimizing the transfer of HDL-derived CE to LDL, ApoF helps control LDL cholesterol levels when LDL clearance mechanisms are limiting.

Site-specific phosphorylation regulates the functions of kindlin-3 in a variety of cells.

Studies of isolated cells, mice, and humans have demonstrated the vital role of the FERM domain protein kindlin-3 in integrin activation in certain hematopoietic and non-hematopoietic cells, consequent to binding to integrin ß-subunits. To explore regulatory mechanisms, we developed a monoclonal antibody that selectively recognizes the phosphorylated form of Ser484 (pS484) in kindlin-3. Activation of platelets, HEL megakaryocytic-like cells and BT549 breast cancer cells led to enhanced expression of pS484 as assessed by immunofluorescence or Western blotting. In platelets, pS484 rose rapidly and transiently upon stimulation. When a mutant form of kindlin-3, T482S484/AA kindlin-3, was transduced into mouse megakaryocytes, it failed to support activation of integrin αIIbß3, whereas wild-type kindlin-3 did. In MDA-MB231 breast cancer cells, expression of T482S484/AA kindlin-3 suppressed cell spreading, migration, invasion, and VEGF production. Wild-type kindlin-3 expressing cells markedly increased tumor growth in vivo, whereas T482S484/AA kindlin-3 significantly blunted tumor progression. Thus, our data establish that a unique phosphorylation event in kindlin-3 regulates its cellular functions.

αMß2 Is Antiatherogenic in Female but Not Male Mice.

Atherosclerosis is a complex inflammatory process characterized by monocyte recruitment into the arterial wall, their differentiation into macrophages, and lipid accumulation. Because integrin αMß2 (CD11b/CD18) mediates multiple diverse functions of leukocytes, we examined its role in atherogenesis. αM-/-/ApoE-/- and ApoE-/- mice were fed a control or high fat diet for 3 or 16 wk to induce atherogenesis. Unexpectedly, αM deficiency accelerated development of atherosclerosis in female but not in male mice. The size of aortic root lesions was 3-4.5-fold larger in female αM-/-/ApoE-/- than in ApoE-/- mice. Monocyte and macrophage content within the lesions was increased 2.5-fold in female αM-/-/ApoE-/- mice due to enhanced proliferation. αMß2 elimination promoted gender-dependent foam cell formation due to enhanced uptake of cholesterol by αM-/-/ApoE-/- macrophages. This difference was attributed to enhanced expression of lipid uptake receptors, CD36 and scavenger receptor A1 (SR-A1), in female mice. Macrophages from female αM-/-/ApoE-/- mice showed dramatically reduced expression of FoxM1 transcription factor and estrogen receptors (ER) α and ß. As their antagonists inhibited the effect of 17ß-estradiol (E2), E2 decreased CD36, SR-A1, and foam cell formation in ApoE-/- macrophages in an ERα- and ERß-dependent manner. However, female αM-/-/ApoE-/- macrophages failed to respond to E2 and maintained elevated CD36, SR-A1, and lipid accumulation. FoxM1 inhibition in ApoE-/- macrophages reduced ERs and enhanced CD36 and SR-A1 expression, whereas FoxM1 overexpression in αM-/-/ApoE-/- macrophages reversed their proatherogenic phenotype. We demonstrate a new, surprising atheroprotective role of αMß2 in female ApoE-/- mice. αMß2 maintains ER expression in macrophages and E2-dependent inhibition of foam cell formation.

Defective triglyceride biosynthesis in CETP-deficient SW872 cells.

We previously reported that reducing the expression of cholesteryl ester transfer protein (CETP) disrupts cholesterol homeostasis in SW872 cells and causes an ∼50% reduction in TG. The causes of this reduced TG content, investigated here, could not be attributed to changes in the differentiation status of CETP-deficient cells, nor was there evidence of endoplasmic reticulum (ER) stress. In short-term studies, the total flux of oleate through the TG biosynthetic pathway was not altered in CETP-deficient cells, although mRNA levels of some pathway enzymes were different. However, the conversion of diglyceride (DG) to TG was impaired. In longer-term studies, newly synthesized TG was not effectively transported to lipid droplets, yet this lipid did not accumulate in the ER, apparently due to elevated lipase activity in this organelle. DG, shown to be a novel CETP substrate, was also inefficiently transferred to lipid droplets. This may reduce TG synthesis on droplets by resident diacylglycerol acyltransferase. Overall, these data suggest that the decreased TG content of CETP-deficient cells arises from the reduced conversion of DG to TG in the ER and/or on the lipid droplet surface, and enhanced TG degradation in the ER due to its ineffective transport from this organelle.

Modification of CETP function by changing its substrate preference: a new paradigm for CETP drug design.

We previously determined that hamster cholesteryl ester transfer protein (CETP), unlike human CETP, promotes a novel one-way transfer of TG from VLDL to HDL, causing HDL to gain lipid. We hypothesize that this nonreciprocal lipid transfer activity arises from the usually high TG/cholesteryl ester (CE) substrate preference of hamster CETP. Consistent with this, we report here that ∼25% of the total lipid transfer promoted by the human Q199A CETP mutant, which prefers TG as substrate, is nonreciprocal transfer. Other human CETP mutants with TG/CE substrate preferences higher or lower than wild-type also possess nonreciprocal lipid transfer activity. Mutants with high TG/CE substrate preference promote the nonreciprocal lipid transfer of TG from VLDL to HDL, but mutants with low TG/CE substrate preference promote the nonreciprocal lipid transfer of CE, not TG, and this lipid flow is in the reverse direction (from HDL to VLDL). Anti-CETP TP2 antibody alters the TG/CE substrate preference of CETP and also changes the extent of nonreciprocal lipid transfer, showing the potential for externally acting agents to modify the transfer properties of CETP. Overall, these data show that the lipid transfer properties of CETP can be manipulated. Function-altering pharmaceuticals may offer a novel approach to modify CETP activity and achieve specific modifications in lipoprotein metabolism.

Overexpression of full-length cholesteryl ester transfer protein in SW872 cells reduces lipid accumulation.

Cells produce two cholesteryl ester transfer protein (CETP) isoforms, full-length and a shorter variant produced by alternative splicing. Blocking synthesis of both isoforms disrupts lipid metabolism and storage. To further define the role of CETP in cellular lipid metabolism, we stably overexpressed full-length CETP in SW872 cells. These CETP(+) cells had several-fold higher intracellular CETP and accumulated 50% less TG due to a 26% decrease in TG synthesis and 2.5-fold higher TG turnover rate. Reduced TG synthesis was due to decreased fatty acid uptake and impaired conversion of diglyceride to TG even though diacylglycerol acyltransferase activity was normal. Sterol-regulatory element binding protein 1 mRNA levels were normal, and although PPARγ expression was reduced, the expression of several of its target genes including adipocyte triglyceride lipase, FASN, and APOE was normal. CETP(+) cells contained smaller lipid droplets, consistent with their higher levels of perilipin protein family (PLIN) 3 compared with PLIN1 and PLIN2. Intracellular CETP was mostly associated with the endoplasmic reticulum, although CETP near lipid droplets poorly colocalized with this membrane. A small pool of CETP resided in the cytoplasm, and a subfraction coisolated with lipid droplets. These data show that overexpression of full-length CETP disrupts lipid homeostasis resulting in the formation of smaller, more metabolically active lipid droplets.

Cholesteryl ester transfer proteins from different species do not have equivalent activities.

Site-specific changes in the amino acid composition of human cholesteryl ester transfer protein (CETP) modify its preference for triglyceride (TG) versus cholesteryl ester (CE) as substrate. CETP homologs are found in many species but little is known about their activity. Here, we examined the lipid transfer properties of CETP species with 80-96% amino acid identity to human CETP. TG/CE transfer ratios for recombinant rabbit, monkey, and hamster CETPs were 1.40-, 1.44-, and 6.08-fold higher than human CETP, respectively. In transfer assays between VLDL and HDL, net transfers of CE into VLDL by human and monkey CETPs were offset by equimolar net transfers of TG toward HDL. For hamster CETP this process was not equimolar but resulted in a net flow of lipid (TG) into HDL. When assayed for the ability to transfer lipid to an acceptor particle lacking CE and TG, monkey and hamster CETPs were most effective, although all CETP species were able to promote this one-way movement of neutral lipid. We conclude that CETPs from human, monkey, rabbit, and hamster are not functionally equivalent. Most unique was hamster CETP, which strongly prefers TG as a substrate and promotes the net flow of lipid from VLDL to HDL.

WAVE3, an actin remodeling protein, is regulated by the metastasis suppressor microRNA, miR-31, during the invasion-metastasis cascade.

WAVE3, an actin cytoskeleton remodeling protein, is highly expressed in advanced stages of breast cancer and influences tumor cell invasion. Loss of miR-31 has been associated with cancer progression and metastasis. Here, we show that the activity of WAVE3 to promote cancer cell invasion is regulated by miR-31. An inverse correlation was demonstrated between expression levels of WAVE3 and miR-31 in invasive versus noninvasive breast cancer cell lines. miR-31 directly targeted the 3'-UTR of the WAVE3 mRNA and inhibited its expression in the invasive cancer cells, i.e., miR-31-mediated down-regulation of WAVE3 resulted in a significant reduction in the invasive phenotype of cancer cells. This relationship was specific to the loss of WAVE3 expression because re-expression of a miR-31-resistant form of WAVE3 reversed miR-31-mediated inhibition of cancer cell invasion. Furthermore, expression of miR-31 correlates inversely with breast cancer progression in humans, where an increase in expression of miR-31 target genes was observed as the tumors progressed to more aggressive forms. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties of cancer cells. The study also identifies a critical role for WAVE3, downstream of miR-31, in the invasion-metastasis cascade.

The integrin co-activator Kindlin-3 is expressed and functional in a non-hematopoietic cell, the endothelial cell.

Integrin activation is crucial for numerous cellular responses, including cell adhesion, migration, and survival. Recent studies in mice have specifically emphasized the vital role of kindlin-3 in integrin activation. Kindlin-3 deficiency in humans also has now been documented and includes symptoms of bleeding, frequent infections, and osteopetrosis, which are consequences of an inability to activate beta1, beta2, and beta3 integrins. To date, kindlin-3 was thought to be restricted to hematopoietic cells. In this article, we demonstrate that kindlin-3 is present in human endothelial cells derived from various anatomical origins. The mRNA and protein for KINDLIN-3 was detected in endothelial cells by reverse transcription-PCR and Western blots. When subjected to sequencing by mass spectrometry, the protein was identified as authentic kindlin-3 and unequivocally distinguished from KINDLIN-1 and KINDLIN-2 or any other known protein. By quantitative real time PCR, the level of kindlin-3 in endothelial cells was 20-50% of that of kindlin-2. Using knockdown approaches, we show that kindlin-3 plays a role in integrin-mediated adhesion of endothelial cells. This function depends upon the integrin and substrate and is distinct from that of kindlin-2. Formation of tube-like structures in Matrigel also was impaired by kindlin-3 knockdown. Mechanistically, the distinct functions of the kindlins can be traced to differences in their subcellular localization in integrin-containing adhesion structures. Thus, the prevailing view that individual kindlins exert their functions in a cell type-specific manner must now be modified to consider distinct functions of the different family members within the same cell type.

Molecular cloning of hamster lipid transfer inhibitor protein (apolipoprotein F) and regulation of its expression by hyperlipidemia.

Lipid transfer inhibitor protein (LTIP) is a regulator of cholesteryl ester transfer protein (CETP) function. Factors affecting plasma LTIP levels are poorly understood. In humans, plasma LTIP is elevated in hypercholesterolemia. To define possible mechanisms by which hyperlipidemia modifies LTIP, we investigated the effects of hypercholesterolemic diets on plasma LTIP and mRNA levels in experimental animals. The hamster, which naturally expresses CETP, was shown to express LTIP. Hamster LTIP mRNA, exclusively detected in the liver, defined a predicted LTIP protein that is 69% homologous to human, with an isoelectric point of 4.15 and Mr = approximately 16.4 kDa. Hyperlipidemia induced by feeding hydrogenated coconut oil, cholesterol, or both lipids increased plasma LTIP mass up to 2.5-fold, with LTIP mass correlating strongly with plasma cholesterol levels. CETP mass was similarly affected by these diets. In contrast, these diets reduced LTIP hepatic mRNA levels by >50%, whereas CETP mRNA was increased. Similar results for both CETP and LTIP were also observed in cholesterol-fed rabbits. In conclusion, we report in hamster and rabbit that dietary lipids regulate LTIP. Diet-induced hypercholesterolemia markedly increased plasma LTIP mass while concomitantly depressing LTIP gene expression. CETP and LTIP have distinct responses to dietary lipids.

Possible role for intracellular cholesteryl ester transfer protein in adipocyte lipid metabolism and storage.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride (TG) between lipoproteins in plasma. However, short term suppression of CETP biosynthesis in cells alters cellular cholesterol homeostasis, demonstrating an intracellular role for CETP as well. The consequences of chronic CETP deficiency in lipid-storing cells normally expressing CETP have not been reported. Here, SW872 adipocytes stably expressing antisense CETP cDNA and synthesizing 20% of normal CETP were created. CETP-deficient cells had 4-fold more CE but an approximately 3-fold decrease in cholesterol biosynthesis. This phenotype of cholesterol overload is consistent with the observed 45% reduction in low density lipoprotein receptor and 2.5-fold increase in ABCA1 levels. However, cholesterol mass in CETP-deficient adipocytes was actually reduced. Strikingly, CETP-deficient adipocytes stored <50% of normal TG, principally reflecting reduced synthesis. The hydrolysis of cellular CE and TG in CETP-deficient cells was reduced by >50%, although hydrolase/lipase activity was increased 3-fold. Notably, the incorporation of recently synthesized CE and TG into lipid storage droplets in CETP-deficient cells was just 40% of control, suggesting that these lipids are inefficiently transported to droplets where the hydrolase/lipase resides. The capacity of cellular CETP to transport CE and TG into storage droplets was directly demonstrated in vitro. Overall, chronic CETP deficiency disrupts lipid homeostasis and compromises the TG storage function of adipocytes. Inefficient CETP-mediated translocation of CE and TG from the endoplasmic reticulum to their site of storage may partially explain these defects. These studies in adipocytic cells strongly support a novel role for CETP in intracellular lipid transport and storage.

AMP-deaminase in elasmobranch fish: a comparative histochemical and enzymatic study.

AMP-deaminase activity was measured in white muscle from a wide range of fish, including one cyclostome, 13 chondrosteans, and one teleost to elucidate the pattern of the AMP-deaminase activity in white muscle of fish. Compared to a mammalian (rat) muscle extract, low enzyme activities are found in the cyclostome and two elasmobranchs from two families (Scyliorhinidae, Hexanchidae). In contrast, higher AMP-deaminase activities, similar to mammals, are expressed in Squalidae, all families of skates, Chimaeridae and in the teleostean fish. We then compared AMP-deaminase activities in red and white muscles from two representative elasmobranch fish, the dogfish (Scyliorhinus canicula) and the thornback ray (Raja clavata). The fibre type composition and distribution of the locomotory musculature were determined in these two elasmobranchs to establish a relationship between the morphology, the type of fibres of the locomotion-implicated muscles and the AMP-deaminase activity. Experimental data are discussed with respect to the layout of fibres in the myotome. In both species, three fibre types were identified. In the two fish myotomes, most of the axial muscles are white fibres while red fibres constitute a thin sheet. Some differences were observed between the two species in the distribution of intermediate fibres: in dogfish, these are located between the red and white fibres; in thornback ray, some are dispersed within the white fibre region, while others form an intermediary layer like in dogfish. These results suggest that in the course of evolution, an amplification of the AMP-deaminase activity in muscle was coupled with increase of complexity of the muscular structure.