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. 2017 Oct 19;2(20):e96783.
doi: 10.1172/jci.insight.96783.

Lipoprotein Lipase Reaches the Capillary Lumen in Chickens Despite an Apparent Absence of GPIHBP1

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Free PMC article

Lipoprotein Lipase Reaches the Capillary Lumen in Chickens Despite an Apparent Absence of GPIHBP1

Cuiwen He et al. JCI Insight. .
Free PMC article

Abstract

In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens - as it does in mammals - despite an apparent absence of GPIHBP1.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Testing the specificity of an immunopurified goat IgG against chicken lipoprotein lipase (cLPL) with Western blots.
Proteins from chicken tissue extracts (30 μg/lane) were size-fractionated by SDS-PAGE and then examined by Western blotting. (A) Western blot with an immunopurified goat IgG against cLPL. The antibodies bound specifically to cLPL from E. coli and recognized a band of similar size in chicken tissues. In heart, the anti-cLPL antibodies also bound to a smaller fragment of approximately 38 kDa. (B) Western blot with the same antibodies after they had been repurified on a second cLPL immunoaffinity column generated with cLPL from E. coli. SKM: skeletal muscle.
Figure 2
Figure 2. Testing the ability of an immunopurified goat IgG against chicken lipoprotein lipase (cLPL) to bind to cLPL in cultured cells.
CHO pgsA-745 cells were transiently transfected with V5-tagged cLPL. Immunocytochemistry studies were performed on fixed and permeabilized cells with a goat antibody against cLPL and a mouse monoclonal antibody against the V5 tag. Binding of the primary antibodies was detected with an Alexa Fluor 555–conjugated donkey anti–goat IgG (red) and an Alexa Fluor 488–conjugated donkey anti–mouse IgG (green). As experimental controls, we included transfected cells that had been incubated with secondary antibodies alone and cells that had been incubated with the anti-cLPL antibody alone. DNA was stained with DAPI (blue). (A) Immunocytochemistry studies on cells that had been fixed with methanol. (B) Studies of cells fixed with paraformaldehyde (PFA). Scale bars: 50 μm.
Figure 3
Figure 3. LPL in chicken tissues is associated with capillaries.
Frozen sections from chicken white adipose tissue (WAT) and heart were stained with a rabbit antibody against von Willebrand factor (vWF), followed by an Alexa Fluor 488–conjugated donkey anti–rabbit IgG (green) and an Alexa Fluor 555–conjugated goat anti–chicken lipoprotein lipase (anti-cLPL) IgG (red). DNA was stained with DAPI (blue). (A) Sections of heart stained with both cLPL and vWF antibodies, revealing colocalization of cLPL and vWF in capillaries. As controls, we examined a section incubated with secondary antibodies alone and a section stained with the vWF antibody and nonimmune goat IgG. (B) An independent chicken immunohistochemistry study in which sections of WAT and heart were stained with cLPL and vWF antibodies. Scale bars: 50 μm.
Figure 4
Figure 4. Chicken lipoprotein lipase (cLPL) is located along the luminal surface of capillaries.
A 9-day-old chicken was given an intravenous injection of 0.7 mg of an Alexa Fluor 555–labeled goat IgG against cLPL (red), 0.5 mg of fluorescein-labeled Lens culinaris agglutinin (Lectin, green), and 0.7 mg of Alexa Fluor 647–labeled nonimmune goat IgG (cyan). After 4 minutes, the chicken was perfused with 50 ml of PBS followed by 30 ml of 3% paraformaldehyde (PFA) in PBS. Liver, white adipose tissue (WAT), heart, and cerebellum were harvested, fixed in 3% PFA, and 50-μm sections of WAT and 10-μm sections of heart, liver, and cerebellum were prepared. The lectin bound to endothelial cells of capillaries and larger blood vessels; the goat IgG against cLPL bound to capillaries but not larger blood vessels (arrows). The nonimmune goat IgG did not bind to blood vessels of the heart, WAT, or cerebellum (indicating an effective perfusion) but as expected did bind to macrophages in the liver. DNA was stained with DAPI (blue). Scale bars: 50 μm.
Figure 5
Figure 5. Chicken lipoprotein lipase (cLPL) can be released from tissues with heparin.
(A) Isolated chicken hearts were perfused with 20 U/ml heparin, and cLPL protein was detected in individual fractions (0.2 ml/fraction) by Western blotting with a goat antibody against cLPL. (B) cLPL activity in the fractions from 3 different chickens (Heparin-1, Heparin-2, Heparin-3) was measured with a [3H]triolein substrate. As a control, a chicken heart was perfused with saline only (Saline). (C) Inhibition of the LPL activity with a goat antiserum against cLPL. For these studies, we pooled fractions 3–5 from 2 of the chickens (Heparin-1 and Heparin-2). We aliquoted 25 μl of the pooled fractions and then added either 50 μl of the goat antiserum against cLPL or normal goat serum. We then performed LPL activity assays. The 4 data points represent duplicate lipase assays on the fractions from 2 chickens. (D) Bar graph showing rapid heparin-mediated release of mouse LPL (mLPL) from an isolated heart of a wild-type mouse (black bars). Heparin-mediated release of mLPL from the heart of a Gpihbp1-deficient mouse was delayed (white bars). Similar results were observed in 2 other pairs of wild-type and Gpihbp1-deficient mice. (E) Rapid release of cLPL (as measured by ELISA) into the plasma of a cockerel after an intravenous injection of heparin (2 U/g body weight). (F) Rapid release of cLPL into the plasma of 4 hens after an intravenous injection of heparin (2 U/g body weight).
Figure 6
Figure 6. Chicken lipoprotein lipase (cLPL) is released from chicken hearts by phosphatidylinositol-specific phospholipase C (PIPLC).
Isolated chicken hearts were perfused with 6 U/ml PIPLC or normal saline alone and incubated in a water bath for 10 minutes at 37°C. The hearts were then perfused with Tyrode’s buffer. Six fractions were collected, and cLPL mass and activity were measured. (A) Western blots demonstrating that PIPLC releases cLPL into the perfusate. (B) Activity assays with a [3H]triolein substrate, revealing that PIPLC releases catalytically active cLPL into the perfusate. Three independent experiments with PIPLC were performed (PIPLC-1, PIPLC-2, PIPLC-3), and 2 studies were performed with normal saline alone (Saline-1, Saline-2). (C) Inhibition of PIPLC-released cLPL activity with a goat antibody against cLPL. We pooled the fractions from the PIPLC-1 and PIPLC-2 experiments, and cLPL activity was measured in the presence of either the goat antiserum against cLPL or normal goat serum. The 4 data points represent lipase assays, performed in duplicate, on the fractions from 2 chickens.
Figure 7
Figure 7. Chicken lipoprotein lipase (cLPL) binds to wild-type mouse GPIHBP1 (mGPIHBP1) or human GPIHBP1 (hGPIHBP1) but not to mutant GPIHBP1 proteins (mGPIHBP1-W108S, hGPIHBP1-W109S) that lack the ability to bind cLPL.
CHO pgsA-745 cells were transiently transfected with S-protein–tagged versions of wild-type or mutant GPIHBP1 and coplated with cells that had been transfected with V5-tagged versions of cLPL. Immunocytochemistry studies were performed on permeabilized and nonpermeabilized cells with a goat antibody against the S-protein tag (red) and a mouse antibody against the V5 tag (green). DNA was stained with DAPI (blue). (A) Immunocytochemistry studies showing that the secreted human (hLPL) or cLPL bound avidly to neighboring CHO cells expressing wild-type hGPIHBP1 (hence, hLPL and cLPL colocalized with hGPIHBP1) but not to CHO cells expressing hGPIHBP1-W109S (no colocalization). (B) Immunocytochemistry studies showing that the secreted mouse LPL (mLPL) or cLPL bound avidly to neighboring CHO cells expressing wild-type mGPIHBP1 (colocalization) but not to CHO cells expressing mGPIHBP1-W108S (no colocalization). Scale bars: 20 μm.
Figure 8
Figure 8. Wild-type chicken lipoprotein lipase (cLPL-wt), but not a mutant cLPL with a p.C420Y mutation, binds to GPIHBP1.
CHO pgsA-745 cells were transiently transfected with S-protein–tagged wild-type human (h) or mouse (m) GPIHBP1 (or hGPIHBP1-W109S or mGPIHBP1-W108S) and coplated with cells that had been transfected with V5-tagged versions of cLPL (wt or C420Y). Immunocytochemistry studies were performed on permeabilized and nonpermeabilized cells with a goat antibody against the S-protein tag (red) and a mouse monoclonal antibody against the V5 tag (green). DNA was stained with DAPI (blue). (A) Immunocytochemistry studies showing that cLPL-wt bound avidly to neighboring CHO cells expressing wild-type hGPIHBP1 (hence cLPL-wt colocalized with hGPIHBP1), whereas cLPL-C420Y had no capacity to bind to cells expressing wild-type hGPIHBP1 (no colocalization). Cells expressing hGPIHBP1-W109S did not bind cLPL-wt. (B) Immunocytochemistry studies showing that cLPL-wt bound avidly to CHO cells expressing wild-type mGPIHBP1 (colocalization), while LPL-C420Y had no capacity to bind to cells expressing wild-type mGPIHBP1 (no colocalization). Cells expressing mGPIHBP1-W108S did not bind cLPL-wt. Scale bars: 20 μm.
Figure 9
Figure 9. Testing the ability of chicken lipoprotein lipase (cLPL) to bind to chicken Ly6E-like proteins on the surface of transfected cells.
CHO pgsA-745 cells were transfected with an expression vector for S-protein–tagged human GPIHBP1 (hGPIHBP1) or for S-protein–tagged versions of the chicken Ly6-like proteins. Immunocytochemistry studies were performed on permeabilized and nonpermeabilized cells with a goat antibody against the S-protein tag (red) and a mouse antibody against the V5 tag (green). DNA was stained with DAPI (blue). (A) Immunocytochemistry studies performed with expression vectors for ENSGALG00000039585, ENSGALG00000043582, and ENSGALG00000041621 showing than none of these chicken Ly6-like proteins bound cLPL (no colocalization). (B) Immunocytochemistry studies performed with 2 different expression vectors for ENSGALG00000045170 (45170a and 45170b). Vector 45170a contained the Ly6 domain of ENSGALG00000045170 and also included the acidic domain of hGPIHBP1; vector 45170b contained the Ly6 domain of ENSGALG00000045170. Neither vector conferred upon CHO cells the ability to bind cLPL. Scale bars: 20 μm.

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