Novel functions of lipid-binding protein 5 in Caenorhabditis elegans fat metabolism

Abstract

The lipid-binding protein (LBP) family is conserved from Caenorhabditis elegans to mammals and essential for fatty acid homeostasis. RNAi-mediated knockdown of nine C. elegans lbp family members revealed that lbp-5 regulates fat accumulation. C. elegans LBP-5 bound directly to various fatty acids with varying affinities. lbp-5 expression in nhr-49(nr2041) worms was much lower than in N2 worms. Nhr-49 transcriptional activity also decreased with lbp-5 deletion, suggesting that they may work together as functional partners in fat metabolism. In support of this notion, LBP-5 translocated into nuclei, where it appeared to influence C. elegans NHR-49 target genes involved in energy metabolism. Interestingly, LBP-5 is required for stearic acid-induced transcription of NHR-49 target genes. Thus, this knowledge could help identify therapeutic targets to treat obesity and diseases associated with nematode-host interactions.

FIGURE 1.

Function of lbp-5 in fat storage. A, Sudan Black staining showed fat accumulation in lbp-5(tm1618), nhr-49(nr2041), and lbp-5(tm1618);nhr-49(nr2041) mutant worms compared with N2 worms. Scale bars, 50 μm. B, triglyceride content for wild-type N2, lbp-5(tm1618), nhr-49(nr2041), and lbp-5(tm1618);nhr-49(nr2041) mutant worms. At least three independent experiments were performed. Error bars indicate the standard deviation. An asterisk indicates a significant difference from the control sample (p < 0.05 as calculated by t test).

FIGURE 2.

Biochemical characterization of recombinant LBP-5 fusion protein. A, CD spectra of purified, E. coli-derived recombinant C. elegans LBP-5 at 0.67 mg/ml in 10 mm potassium phosphate at pH 7.4 and ambient temperature. B, titration curves for the binding of DAUDA to LBP-5. Fixed amounts (1 μm) of LBP-5 were incubated with 0–10 μm DAUDA. Fluorescence was measured (excitation 345 nm, emission 420–600 nm) after equilibration. C, binding of DAUDA to recombinant LBP-5 and competition with stearic acid. Fluorescence emission spectra (excitation at 345 nm) of 1 μm DAUDA alone or together with 1 μm LBP-5 monomer are shown. Also shown is the reversal of changes in DAUDA emission by competition with stearic acid (0.5, 1, 2, 4, and 8 μm) added to the LBP-5-DAUDA complex (both 1 μm). At least three independent experiments were performed. D, Scatchard analysis of DAUDA binding to purified C. elegans LBP-5. The data in panel B were subjected to Scatchard analysis. The maximal fluorescence achieved in the presence of excess DAUDA was assumed to represent 100% binding. Moreover, the amount of DAUDA bound was assumed to be proportional to the relative intensity. At low molar DAUDA/LBP-5 ratios (up to ∼0.7), a binding site with an apparent K_d of 0.033 μm (B_max = 0.7; (b)) was identified. At higher molar ratios, a single binding site with apparent K_d of 0.48 μm (B_max = 1.1) was identified (a). Correlation coefficients: line a, r = 0.98; line b, r = 0.99.

FIGURE 3.

Spatio-temporal expression pattern of LBP-5-GFP in C. elegans. A, expression in the intestine and nuclei in an L3 larva. B, punctate fluorescence seen in adult seam cells. C, vulva expression in an adult. D, expression in seam and intestine of an L4 larva. E, hypodermal expression in an L4 larva. F, hypodermal expression in an egg. Scale bars, 50 μm.

FIGURE 4.

Regulation of lbp-5 expression by nhr-49. A, expression level of lbp-5 in nhr-49 mutants (L4) by qRT-PCR. Actin served as an internal control. Three independent experiments were performed. Error bars indicate the standard deviation. B, changes in fluorescence intensity of adult worms after nhr-49-specific RNAi feeding to lbp-5-gfp worms. Images were acquired with identical exposure times. Scale bars, 100 μm.

FIGURE 5.

A summary diagram showing the regulatory effects of NHR-49. A, the different boxes represent genes involved in C. elegans mitochondrial β-oxidation, peroxisomal β-oxidation, fatty acid desaturation/elongation, and gluconeogenesis. qRT-PCR was used to measure the expression of these NHR-49 target genes in N2 and nhr-49(nr2041) worms (see supplemental Fig. S7). The red dashed line represents down-regulation and the green arrowhead represents up-regulation. B, logarithmic plot of the qRT-PCR gene expression levels of NHR-49 target genes involved in fatty acid desaturation/elongation in N2, lbp-5(tm1618), nhr-49(nr2041), and lbp-5(tm1618);nhr-49(nr2041) worms fed with stearic acid. A detailed graph showing the actual relative expression level of NHR-49 targets involved in fatty acid desaturation/elongation in N2 worms fed with stearic acid compared with control. C, gene expression levels of NHR-49 targets involved in gluconeogenesis in N2, lbp-5(tm1618), nhr-49(nr2041), and lbp-5(tm1618);nhr-49(nr2041) worms fed with stearic acid as assessed by qRT-PCR. Actin served as an internal control. At least three independent experiments were performed. Error bars indicate the standard deviation.

FIGURE 6.

A proposed model of coordination between LBP-5 and NHR-49. LBP-5 transports fatty acids into the nucleus and affects gene expression. LBP-5 as a cofactor of NHR-49 may regulate its functions in energy metabolism. Stearic acid (SA) is a specific NHR-49 activator. LBP-5 may transport SA from the cytoplasm to the nucleus where NHR-49 is present, which results in promotion of fatty acid desaturation/elongation by activating fat-5, -6, and -7 expression. LBP-5 may also enhance fatty acid gluconeogenesis by activating gei-7 and sdha-2 expression. NHR-49 may enhance lbp-5 expression to promote fatty acid transport. However, this coordinated effect of LBP-5 and NHR-49 on the mitochondrial and peroxisomal β-oxidation enzymes remains to be determined. Fatty acid desaturation/elongation: Δ-9 fatty acid desaturase (fat-5, fat-6, fat-7), gluconeogenesis:isocitrate lyase/malate synthase (gei-7), and succinate dehydrogenase complex (sdha-2) are indicated.