Endothelial lipase provides an alternative pathway for FFA uptake in lipoprotein lipase-deficient mouse adipose tissue

Abstract

Lipoprotein lipase (LPL) is thought to be the only enzyme responsible for the catabolism of triglycerides (TGs) associated with TG-rich lipoproteins in adipose tissue (AT). However, LPL deficiency in humans and induced mutant mice is not associated with decreased fat mass. We investigated whether endothelial lipase (EL), a recently discovered phospholipase, might represent an alternative mechanism for the uptake of phospholipid-derived fatty acids in murine lipoprotein-deficient AT. When LPL was expressed in AT and isolated murine adipocytes, EL mRNA was not detectable. In contrast, mouse AT and isolated adipocytes that lacked LPL expressed large amounts of EL mRNA. The cellular phospholipase activity in LPL-deficient fat pads was increased 4-fold compared with control fat pads and could be inhibited to control levels by a specific EL antibody. Fatty acids produced by EL activity were absorbed by adipocytes and incorporated into the TG moiety of AT. Our results suggest that EL activity in AT and other peripheral tissues might contribute to the tissue uptake of free fatty acids, which could have important implications for the metabolism of plasma lipoproteins.

Figure 1

Murine LPL and EL mRNA expression in adipose tissue. Relative abundance of (A) LPL and (B) EL mRNA in AT of mice expressing (L2, L2-MCK) or lacking (L0-MCK, L0) LPL in AT. Total RNA was isolated from murine AT in the absence or presence of LPL and subjected to Northern blot analysis. Ten micrograms of total RNA were separated on a formaldehyde/agarose gel, blotted onto a nylon membrane, and hybridized with a murine-specific [³²P]-labeled cDNA probe. rRNA, ribosomal RNA. (C) EL mRNA concentrations in AT were confirmed by fluorescent real-time PCR (see Methods). EL mRNA quantities were normalized to those of β-actin. Data are expressed as the mean values of 3 experiments performed in duplicates relative to the expression of EL mRNA in control mice (L2). Bars represent mean ± SD of the hybridization intensity signals. *P < 0.05.

Figure 2

Murine EL mRNA expression in liver. Relative abundance of EL mRNA in livers in the presence (L2) or absence (L0-MCK) of AT-LPL were compared with EL expression in AT of L0-MCK mice. Total RNA was isolated from fat pads of fed L2 and L0-MCK mice. mRNA concentrations were determined by fluorescent real-time PCR. EL mRNA quantities were normalized to those of β-actin. Data are expressed as the mean values of 3 experiments performed in duplicate. Bars represent mean ± SD of the hybridization intensity signals. Inset: Representative Northern blot analysis of 10 μg total RNA per lane, hybridized with a murine-specific [³²P]-labeled EL cDNA probe. Lane 1, L0-MCK AT; lane 2, L2 liver; lane 3, L0-MCK liver.

Figure 3

LPL and EL mRNA expression in differentiated 3T3-L1 cells and isolated mouse adipocytes. Fat pads of mice in the presence (L2) or absence (L0-MCK) of LPL were homogenized and subjected to collagenase A digestion, and total RNA of adipocytes was isolated. Total RNA from 3T3-L1 fibroblasts was isolated at 50% cell confluence (day –2), at confluence (day 0) and during (days 2, 4, 6, and 7), and at the end of differentiation (day 8) to adipocytes. (A) Northern blot analysis of 10 μg total RNA per lane, hybridized with a murine-specific [³²P]-labeled LPL probe. (B) EL mRNA concentrations were determined by fluorescent real-time PCR. EL mRNA quantities were normalized to those of β-actin, and control values from L2 mice were arbitrarily set to 1. *P < 0.05.

Figure 4

Phospholipase activity in murine fat pads. Fat pads of mice in the presence (L2) or absence (L0-MCK) of LPL were incubated in 250 μl DMEM/2% BSA and 2 U/ml heparin for 1 hour at 37–C. Phospholipase activity was measured by incubating the preconditioned medium with [¹⁴C]PC in the absence (control) or presence (EL Ab) of 50 μg anti-EL IgG. Results are mean ± SD from 5 mice of each genotype performed in duplicate. **P – 0.01.

Figure 5

Uptake and incorporation of PL-derived FFAs by AT. Fat pads of mice in the presence (L2) or absence (L0-MCK) of LPL were incubated with DMEM/10% lipoprotein-deficient serum (LPDS) containing [¹⁴C]PC-labeled HDL (50 μg HDL-associated protein/sample) at 37°C for 6 hours. The incorporation of FFAs into the TG moiety of AT was performed in the absence (control) or presence of 50 μg anti-EL IgG, 50 μg/ml THL or/and 2 U/ml heparin in a total volume of 150 μl. Results are mean ± SD from 5 mice of each genotype performed in duplicate. **P – 0.01.

Figure 6

Uptake and incorporation of albumin-bound FFAs by AT. Fat pads of mice in the presence (L2) or absence (L0-MCK) of LPL were incubated with DMEM/1% FFA-free BSA/200 μM oleate/[³H]oleate (5 μCi/ml) at 37–C for 6 hours. Results are mean ± SD from 3 mice of each genotype performed in triplicate. ***P – 0.001.