Unusual metabolic characteristics in skeletal muscles of transgenic rabbits for human lipoprotein lipase

Abstract

Background: The lipoprotein lipase (LPL) hydrolyses circulating triacylglycerol-rich lipoproteins. Thereby, LPL acts as a metabolic gate-keeper for fatty acids partitioning between adipose tissue for storage and skeletal muscle primarily for energy use. Transgenic mice that markedly over-express LPL exclusively in muscle, show increases not only in LPL activity, but also in oxidative enzyme activities and in number of mitochondria, together with an impaired glucose tolerance. However, the role of LPL in intracellular nutrient pathways remains uncertain. To examine differences in muscle nutrient uptake and fatty acid oxidative pattern, transgenic rabbits harboring a DNA fragment of the human LPL gene (hLPL) and their wild-type littermates were compared for two muscles of different metabolic type, and for perirenal fat.

Results: Analyses of skeletal muscles and adipose tissue showed the expression of the hLPL DNA fragment in tissues of the hLPL group only. Unexpectedly, the activity level of LPL in both tissues was similar in the two groups. Nevertheless, mitochondrial fatty acid oxidation rate, measured ex vivo using [1-(14C)]oleate as substrate, was lower in hLPL rabbits than in wild-type rabbits for the two muscles under study. Both insulin-sensitive glucose transporter GLUT4 and muscle fatty acid binding protein (H-FABP) contents were higher in hLPL rabbits than in wild-type littermates for the pure oxidative semimembranosus proprius muscle, but differences between groups did not reach significance when considering the fast-twitch glycolytic longissimus muscle. Variations in both glucose uptake potential, intra-cytoplasmic binding of fatty acids, and lipid oxidation rate observed in hLPL rabbits compared with their wild-type littermates, were not followed by any modifications in tissue lipid content, body fat, and plasma levels in energy-yielding metabolites.

Conclusions: Expression of intracellular binding proteins for both fatty acids and glucose, and their following oxidation rates in skeletal muscles of hLPL rabbits were not fully consistent with the physiology rules. The modifications observed in muscle metabolic properties might not be directly associated with any LPL-linked pathways, but resulted likely of transgene random insertion into rabbit organism close to any regulatory genes. Our findings enlighten the risks for undesirable phenotypic modifications in micro-injected animals and difficulties of biotechnology in mammals larger than mice.

Figure 1

Expression of human lipoprotein lipase (hLPL) mRNA in transgenic rabbits. The cDNA obtained by reverse transcription (RT) of total RNA extracted from skeletal muscles or perirenal fat and primed by random primers followed by 35 cycles of PCR with hLPL-specific primers, was loaded on 2% agarose gel. Reaction was performed in parallel in the absence of reverse transcriptase (RT-), to ensure for lack of genomic DNA contamination. Typical RT-PCR results are shown for semimembranosus proprius muscle. Lanes 1–4: RT-PCR product in hLPL rabbit; Lane 5: RT- in hLPL rabbit; Lane 6: 100 bp DNA ladder; Lane 7–10: RT-PCR product in wild-type rabbit; Lane 11: RT- in wild-type rabbit. A band at the expected size of 137 bp was detected in hLPL rabbits only.

Figure 2

Mitochondrial and peroxisomal oxidation rates of oleate. Oxidation rates were measured in freshly excised samples of semimembranosus proprius and longissimus muscles, using [1-¹⁴C]oleate as substrate in the presence (peroxisomal oxidation) or absence (total oxidation) of mitochondrial inhibitors. Mitochondrial oxidation rates were calculated by difference between total and peroxisomal oxidation rates. Values shown are mean ± SEM of oleate oxidation (nmole/min^-1 per g of muscle wet weight). The * indicates a significant difference (P < 0.05) in mitochondrial oxidation rate in hLPL rabbits in comparison with their wild-type (WT) littermates.