J.H. Veerkamp

Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss

A number of biochemical defects have been identified in glucose metabolism within skeletal muscle in obesity, and positive effects of weight loss on insulin resistance are also well established. Less is known about the capacity of skeletal muscle for the metabolism of fatty acids in obesity‐related insulin resistance and of the effects of weight loss, though it is evident that muscle contains increased triglyceride. The current study was therefore undertaken to profile markers of human skeletal muscle for fatty acid metabolism in relation to obesity, in relation to the phenotype of insulin‐resistant glucose metabolism, and to examine the effects of weight loss. Fifty‐five men and women, lean and obese, with normal glucose tolerance underwent percutaneous biopsy of vastus lateralis skeletal muscle for determination of HADH, CPT, heparin‐releasable (Hr) and tissue‐extractable (Ext) LPL, CS, COX, PFK, and GAPDH enzyme activities, and content of cytosolic and plasma membrane FABP. Insulin sensitivity was measured using the euglycemic clamp method. DEXA was used to measure FM and FFM. In skeletal muscle of obese individuals, CPT, CS, and COX activities were lower while, conversely, they had a higher or similar content of FABPC and FABPPM than in lean individuals. Hr and Ext LPL activities were similar in both groups. In multivariate and simple regression analyses, there were significant correlations between insulin resistance and several markers of FA metabolism, notably, CPT and FABPPM. These data suggest that in obesity‐related insulin resistance, the metabolic capacity of skeletal muscle appears to be organized toward fat esterification rather than oxidation and that dietary‐induced weight loss does not correct this disposition.—Simoneau, J.‐A., Veerkamp, J. H., Turcotte, L. P., Kelley, D. E. Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss. FASEB J. 13, 2051–2060 (1999)

Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate.

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked using dehydrothermal (DHT) treatment and/or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), in the presence and absence of chondroitin sulphate (CS). EDC covalently attaches CS to collagen. DHT crosslinking preserved a porous matrix structure. However, attachment of CS to DHT-treated matrices using EDC, resulted in collapsed surfaces, CS located only at the matrix exterior. EDC crosslinking resulted in a partial matrix collapse. This could be prevented if crosslinking was carried out in the presence of ethanol. Matrix porosity was then preserved. The presence of CS during EDC crosslinking resulted in covalent immobilization of CS throughout the matrix. The amount of CS incorporated was increased if crosslinking was performed in the presence of ethanol. EDC-crosslinked matrices, with and without CS, had increased denaturation temperatures and decreased free amine group contents. The susceptibility of these matrices towards degradation by proteolytic enzymes was diminished. Immobilized CS increased the water-binding capacity and decreased the denaturation temperature and tensile strength. Immobilized CS bound anti-CS antibodies and was susceptible to chondroitinase ABC digestion, demonstrating its bioavailability. The modified matrices were not cytotoxic as was established using human myoblast and fibroblast culture systems. It is concluded that the use of ethanol during EDC crosslinking, offers an elegant means for the preparation of defined porous collagenous matrices containing bioavailable, covalently attached CS.

Differential investigation of the capacity of succinate oxidation in human skeletal muscle.

Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6-dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder.

Structural and functional features of different types of cytoplasmic fatty acid-binding proteins

Article de synthese sur les donnees recentes de caracteristiques structurales et physicochimiques de divers types de proteines de liaison aux acides gras, avec la signification physiologique de ces diversites

Development of tailor-made collagen-glycosaminoglycan matrices: EDC/NHS crosslinking, and ultrastructural aspects

The many biocharacteristics of glycosaminoglycans (GAGs) make them valuable molecules to be incorporated in collagenous biomaterials. To prepare tailor-made collagen-GAG matrices with a well-defined biodegradability and (bioavailable) GAG content, the crosslinking conditions have to be controlled. Additionally, the ultrastructural location of GAGs in engineered substrates should resemble that of the application site. Using chondroitin sulfate (CS) as a model GAG, these aspects were evaluated. The methodology was then applied for other GAGs. CS was covalently attached to collagen using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A maximum of about 155 mg CS/g matrix could be immobilized. CS incorporation and bioavailability, as evaluated by interaction with specific antibodies and glycosidases, was dependent on the molar ratio EDC:carboxylic groups of CS. The denaturation temperature could be modulated from 61 to 85 degrees C. The general applicability of EDC/NHS for immobilizing GAGs was demonstrated with dermatan sulfate, heparin, and heparan sulfate. These matrices revealed comparable physico-chemical characteristics, biodegradabilities, and preserved bioavailable GAG moieties. At the ultrastructural level, GAGs appeared as discrete, electron-dense filaments, each filament representing a single GAG molecule. Distribution was independent of GAG type. They were observed throughout the matrix fibers and at the outer sites, and located, either parallel or orthogonally, at the periphery of individual collagen fibrils. Compositional and ultrastructural similarity between matrices and tissue structures like cartilage and basement membranes can be realized after attachment of specific GAG types. It is concluded that EDC/NHS is generally applicable for attachment of GAGs to collagen. Modulation of crosslinking conditions provides matrices with well-defined GAG contents, and biodegradabilities. Ultrastructural similarities between artificially engineered scaffolds and their possible application site may favor the use of specific collagen-GAG matrices in tissue engineering.

Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) -->/<-- ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK-PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.

Generation and Application of Type-specific Anti-Heparan Sulfate Antibodies Using Phage Display Technology FURTHER EVIDENCE FOR HEPARAN SULFATE HETEROGENEITY IN THE KIDNEY

Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A “single pot” semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene,DP-42), and YYHYKVN (VH1 gene,DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. K d values for HS are about 0.1 μm. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- andN-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.

A radiochemical procedure for the assay of fatty acid binding by proteins

Protein-bound and unbound fatty acids can be efficiently separated at 0 degree C using a hydrophobic column-packing material (Lipidex 1000) similar to the separation of protein-bound and unbound steroids (E. Dahlberg, M. Snochowski, and J.-A. Gustafsson (1980) Anal Biochem. 106, 380-388). Protein-bound fatty acids are also removed by Lipidex 1000 when treatment is performed at 37 degrees C. Lipidex 1000 does not exhibit binding properties for soluble proteins at 0 and 37 degrees C, in contrast to dextran-coated charcoal. Lipidex 1000 appeared to be useful for the delipidation of protein samples at 37 degrees C and for a radiochemical assay of fatty acid-binding by microgram amounts of protein at 0 degree C. With this assay we obtained results on palmitate binding to serum albumin similar to those reported on the basis of equilibrium dialysis. Delipidated proteins from dealbuminized rat liver cytosol maximally bind about 4 nmol palmitate/mg protein.

Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro

An increasing amount of interest is focused on the potential use of tissue-engineered articular cartilage implants, for repair of defects in the joint surface. In this perspective, various biodegradable scaffolds have been evaluated as a vehicle to deliver chondrocytes into a cartilage defect. This cell-matrix implant should eventually promote regeneration of the traumatized articular joint surface with hyaline cartilage. Successful regeneration can only be achieved with such a tissue-engineered cartilage implant if the seeded cells reveal an appropriate proliferation rate in the biodegradable scaffold together with the production of a new cartilage-specific extracellular matrix. These metabolic parameters can be influenced by the biochemical composition of a cell-delivery scaffold. Further elucidation of specific cell-matrix interactions is important to define the optimal biochemical composition of a cell-delivery vehicle for cartilage repair. In this in vitro study, we investigated the effect of the presence of cartilage-specific glycosaminoglycans in a type I collagen scaffold on the metabolic activity of seeded chondrocytes. Isolated bovine chondrocytes were cultured in porous type I collagen matrices in the presence and absence of covalently attached chondroitin sulfate (CS) up to 14 days. CS did indeed influence the bioactivity of the seeded chondrocytes. Cell proliferation and the total amount of proteoglycans retained in the matrix, were significantly higher (p < 0.001) in type I collagen scaffolds with CS. Light microscopy showed the formation of a more dense cartilaginous layer at the matrix periphery. Scanning electron microscopy revealed an almost complete surfacing of the initially porous surface of both matrices. Histology and reverse transcriptase PCR for various proteoglycan subtypes suggested a good preservation of the chondrocytic phenotype of the seeded cells during culture. The stimulatory potential of CS on both the cell-proliferation and matrix retention, turns this GAG into an interesting biochemical component of a cell-delivery scaffold for use in tissue-engineering articular cartilage.

A mitochondrial encephalomyopathy: the first case with an established defect at the level of coenzyme Q

A patient is presented who had therapy-resistant epileptic seizures from the 7th day of life. Examination at the age of 17 months revealed a mentally retarded boy with epileptic seizures, generalised myoclonic contractions, and abnormal ocular movements. A cerebral CT scan showed central and cortical atrophy. Lactate levels in serum, cerebrospinal fluid and urine were elevated, the pyruvate level was raised in serum. A quadriceps muscle biopsy revealed aspecific morphologic signs of a myopathy. Biochemical analysis showed decreased substrate oxidation rates in the mitochondria associated with low rates of ATP production. Total and free carnitine levels were decreased. Investigation of the respiratory chain revealed a defect in the proximal part of respiratory chain revealed a defect in the proximal part of respiratory chain involving the region of coenzyme Q. Based on clinical and chemical data it is likely that the patient is suffering from a multi-system disorder.