Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo

Abstract

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.

Figure 1

Generation of mice expressing inactive hLPL in muscle. (A) hLPL minigene with MCK promoter, cDNA (box), and intron 3. The catalytic triad and the mutation are marked. (B) Genomic screening by using a TaqI polymorphism. (C) Reverse transcription–PCR showing expression of hLPL in skeletal (Mu) and less expression in heart (H) muscle. Li, liver; AT, adipose tissue.

Figure 2

VLDL turnover study. VLDL was labeled with [¹⁴C]palmitate to observe triglyceride hydrolysis (A), with [³H]cholesteryl oleyl ether as a marker for VLDL core lipids (B) and with [¹²⁵I]-tyramine cellobiose apo B for lipoprotein particle uptake (C). The experiments were done on LPL2 (not shown in B and C) and on LPL1 background. Plasma radioactivity in percentage of 0-min value was calculated for each animal. Computer generated curves using a two pool decay model were added. ∗, P < 0.05 vs. LPL1.

Figure 3

VLDL organ uptake study. The experiment was done as in Fig. 2. Organ uptake of palmitic acid was derived from VLDL-TG (A), VLDL cholesteryl ether (B), and VLDL particle (C) uptake. The mean radioactivity for each organ of the LPL2 animals was set to 100%. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 each vs. LPL2 or LPL1, respectively.

Figure 4

Muscle histology. Expression of inactive LPL in muscle leads to a fatty acid-induced mitochondriopathy. The femoral muscles of 6-month-old wild-type (A and C) and high inactive LPL-expressing mice (B and D) are shown. (A and B) Glycogen staining (periodic acid Schiff, ×113). (C and D) Semithin sections (azur-methylene-blue, ×113). Red arrows, subsarcolemnal glycogen (reddish); green arrowheads, examples for centralized nuclei; yellow arrows, dark-blue stained mitochondria.