Silkworm haemolymph induced both the cessation of growth and an increase in triglyceride (triacylglycerol) storage in BmN4 cells. We purified the growth inhibitory factor from the silkworm haemolymph and identified this protein as the Bombyx mori PP (promoting protein), an orthologue of NPC2 (Niemann–Pick disease type C2) protein. Recombinant silkworm NPC2 inhibited cellular proliferation and increased triglyceride accumulation in BmN4 cells. Injection of either the recombinant protein or antiserum of NPC2 into living silkworms increased or decreased respectively triglyceride levels in the fat body. A mutation that depletes the cholesterol-binding capacity did not abolish the activity of NPC2. We further revealed that NPC2 induced the phosphorylation of AMPK (AMP-activated protein kinase) and that an AMPK inhibitor suppressed NPC2-dependent triglyceride accumulation. These findings suggest that NPC2 induces triglyceride accumulation via the activation of AMPK independently of its cholesterol-binding capacity in the silkworm.

INTRODUCTION

Lipid metabolism is regulated by factors in the blood or body fluids in connection with food consumption and developmental stage. These factors, such as insulin, are widely conserved among various organisms [1]; the insulin pathway has been intensively studied in mice, flies and nematodes. Defects in these extracellular factors or associated pathways cause abnormal lipid profiles and sometimes result in metabolic diseases. For example, dysregulation of leptin leads to obesity in mice and humans [2]. Therefore elucidation of humoral factors involved in the lipid metabolic system would deepen our understanding of lipid metabolism and potentially provide insight into metabolic diseases.

NPC (Niemann–Pick disease type C) is a fatal neurodegenerative disease characterized by the abnormal accumulation of cholesterol and glycolipids [3]. Mutations in the NPC1 and NPC2 genes are responsible for 95% and 5% of NPC cases respectively. NPC2 protein is an abundant component of human epididymal fluid, whereas NPC1 protein is located in intracellular membranes [4,5]. Knockout of the Npc2 gene in mice and Drosophila causes abnormal development of cerebellar Purkinje cells and whole brains respectively, indicating the conserved importance of NPC2 in neural development [6,7]. Biochemical studies revealed that NPC2 protein is involved in the regulation of haemopoiesis and immune responses [8,9]. These findings suggest that NPC2 protein plays a crucial role in altering cell fate and function.

NPC2 protein is a highly conserved cholesterol-binding protein [10,11]. NPC2 protein exists in lysosomes and is partially secreted. NPC2 protein can directly transport cholesterol in endolysosome compartments, and intracellular NPC2 protein may play an important role in cholesterol homoeostasis [5,12,13]. Extracellular NPC2 protein modulates haemopoietic differentiation independent of its cholesterol-binding capacity [8]. Therefore NPC2 protein may act not only as a cholesterol carrier, but also as an extracellular signalling molecule. A previous study demonstrated that knockdown of the NPC2 gene in mature adipocytes suppresses the storage of triglyceride, a major energy source lipid [14]. Phosphorylation cascades, such as the AMPK (AMP-activated protein kinase) pathway, downstream of extracellular factors are important for regulating cellular triglyceride (triacylglycerol) levels [1517]. These results imply that extracellular NPC2 protein affects triglyceride accumulation. The biochemical activities of secreted NPC2 protein, however, are not fully understood.

In the present study, we identified Bombyx mori PP (promoting protein), an orthologue of NPC2 protein [18], as a triglyceride accumulation factor. Furthermore, we demonstrated that the promoting effect of extracellular NPC2 protein on triglyceride accumulation was independent of its cholesterol-binding capacity. These findings reveal a new aspect of NPC2 protein as an autocrine/paracrine factor.

EXPERIMENTAL

Insect and reagents

Silkworm eggs (Hu•Yo×Tukuba•Ne) were purchased from Ehime Sanshu. Silkworm larvae were reared on an antibiotic-free artificial diet at 27°C. TC-100 insect culture medium was purchased from AppliChem. FBS and calf serum were purchased from HyClone Laboratories and SAFC Biosciences respectively. Gentamicin, proteinase K and dexamethasone were purchased from Wako Pure Chemicals. Amphotericin B was purchased from Bristol–Myers. Sf-900 II serum-free medium, DMEM (Dulbecco's modified Eagle's medium; high glucose) and penicillin/streptomycin were purchased from Invitrogen. Compound C was purchased from Calbiochem. [3H]Thymidine, Mono S and Superose 12 columns were obtained from GE Healthcare. Pronase E, mannose 6-phosphate and IBMX (isobutylmethylxanthine) were purchased from Sigma–Aldrich. Heparin Toyopearl and Butyl Toyopearl resin were purchased from TOSOH.

Antibodies

Anti-His tag antibody was purchased from Santa Cruz Biotechnology (H-15; catalogue number SC-803). Anti-silkworm NPC2 antiserum was prepared by immunizing a Japanese white rabbit with an intracutaneous injection of a mixture of the recombinant protein and Freund's adjuvant five times at 2-week intervals (T. K. Craft). Primary rabbit antibodies against total AMPK (catalogue number 2532S), phospho-AMPK (catalogue number 2531S), total ERK (extracellular-signal-regulated kinase; catalogue number 4695S), phospho-ERK (catalogue number 4370S) and β-actin (catalogue number 4967S) were obtained from Cell Signaling Technology Japan. HRP (horseradish peroxidase)-linked whole antibody anti-(rabbit IgG) (from donkey; catalogue number NA934) was purchased from GE Healthcare.

Preparation of silkworm haemolymph

Abdominal legs of silkworm larvae (day 5 or 6 of 5th instar) were cut with scissors, and the blood from the wound was collected in ice-cold tubes containing 100 μM 1-phenyl-2-thiourea. Silkworm blood was centrifuged at 10000 g for 10 min and the supernatant (plasma) was frozen in liquid nitrogen. Samples were stored at −20°C before use. The plasma fraction was further incubated at 60°C for 30 min followed by centrifugation. The obtained supernatant is hereafter referred to as the haemolymph fraction.

Cell culture

Silkworm-derived BmN4 cells were cultured in a tissue culture flask (catalogue number 3100-25 and 3110-75; IWAKI) containing TC-100 medium supplied with 10% FBS at 27°C. Sf-9 cells were purchased from Novagen and maintained in Sf-900 II serum-free medium at 27°C. To test the effects of the haemolymph and purified fractions, 50 μg/ml gentamicin, 2.5 μg/ml amphotericin B and 10% (v/v) of test samples were also added to the above-described medium. When test samples comprised less than 10% of the total volume, 0.9% NaCl (saline) was added. 3T3-L1 cells were purchased from the JCRB Cell Bank (Tokyo, Japan; #JCRB9014) and maintained at 37°C with 5% CO2 in DMEM with 10% calf serum, penicillin (100 IU) and streptomycin (100 μg/ml). CHO-K1 cells were kindly provided by Dr Makoto Arita and Dr Hiroyuki Arai (The University of Tokyo), and were maintained at 37°C with 5% CO2 in Ham's F12 medium (Wako Pure Chemicals) with 10% FBS.

Measurement of cell growth

BmN4 cells were collected using cell scrapers and viable cell numbers were counted by a cytometer after Trypan Blue staining. After a 24-h incubation, the culture medium was replaced with test samples. At each time point, cells were collected and counted as described above.

Thymidine incorporation assay

BmN4-cell suspension (500 μl; 2×105 cells/ml) was applied to each well of a 24-well tissue culture plate. After a 24-h incubation in culture medium containing the test samples, cultures were supplied with 0.5 μCi/well [3H]thymidine and further incubated for 6 h. Cells were washed with PBS and then lysed in 500 μl of lysis buffer containing 10 mM Tris/HCl (pH 8.0), 1 mM EDTA and 2% SDS. Lysates were mixed with 55 μl of 100% TCA (trichloroacetic acid) in ice-cold tubes, and insoluble fractions absorbed on glass filters were obtained by filtration under reduced pressure. Filters were washed twice with a buffer containing 5% TCA and 5 g/l sodium pyrophosphate, followed by drying under an infrared lamp. Filters were then soaked in toluene containing 4 g/l 2,5-diphenyloxazone, and radioactivity was measured by a liquid scintillation counter (Beckman Coulter). Inhibition of thymidine incorporation was determined as a decrease in the radioactivity of the test samples relative to those prepared from cultures supplied with saline (10%, v/v).

Protease treatment

The haemolymph fraction was incubated with 50 μg/ml proteinase K and 100 μg/ml pronase E at 37°C. After 16 h, the haemolymph was centrifuged at 15000 g for 1 min, and the supernatant was used as a protease-treated sample. For the mock-treated sample, Milli-Q water was added to the haemolymph fraction and the mixture was incubated as described above.

Purification and identification of [3H]thymidine incorporation-inhibitory factor

One unit is defined as the amount of activity that inhibited 30% of [3H]thymidine incorporation into BmN4 cells. For details regarding the purification processes and production of recombinant proteins, see the Supplementary Online Data (http://www.biochemj.org/bj/459/bj4590137add.htm).

Cholesterol-binding assay

The cholesterol-binding assay was performed according to a previous report [13] with modifications. Each recombinant protein (50 μg) was incubated in a labelling solution [10 mM sodium phosphate (pH 7.0), 130 mM NaCl and 0.01% Triton X-100] supplied with different concentrations of [3H]cholesterol for 30 min at 30°C. Free and bound cholesterol were separated by centrifugation through a CentriSep gel filtration column at 750 g for 2 min. Radioactivity of the passed-through fractions was measured with a liquid scintillation counter.

Triglyceride quantification and Oil Red O staining in BmN4 cells

BmN4-cell suspension (8 ml; 2×105 cells/ml) was applied to a 25-cm2 tissue culture plate. After 6 h, the medium was replaced with test samples, and the cells were incubated for another 48 h. Cells were then washed with PBS and collected using cell scrapers. Lipids were extracted by the Bligh–Dyer method, and triglyceride amounts were measured by the GPO (glycerol-3-phosphate oxidase)-DAOS [N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt] method with the Triglyceride E test (Wako Pure Chemicals) according to the manufacturer's instructions. Accumulated quantities of triglyceride were determined by measuring the triglyceride/cell number ratio. Oil Red O staining was performed according to a previous report [19] with modifications. Briefly, cells were fixed with 10% formaldehyde and stained with Oil Red O in 60% propan-2-ol for 10 min at room temperature (25°C).

Triglyceride quantification in silkworm fat body tissues

Female larvae were selected on day 1 of the 5th instar and reared on an artificial diet. Four silkworms on day 2 of the 5th instar with a mass of 1.9±0.2 g/larva were injected with 50 μl of either buffer [25 mM Hepes (pH 7.2), 20% (w/v) glycerol and 100 mM NaCl] or recombinant NPC2 (300 μg/ml). After 24 h, fat body tissues were dissected and homogenized. Lipid extraction and triglyceride quantification were performed as described above. In addition, DNA contents of the fat body tissues were measured from A280 values of samples obtained by PCI (phenol-chloroform-isoamyl alcohol) extraction followed by ethanol precipitation. Accumulated quantities of triglyceride in silkworms injected with NPC2 relative to the control group were determined by comparing the triglyceride/DNA ratios.

qPCR (quantitative real-time PCR) analysis

Total RNA was extracted from cells and fat body lysates using an RNeasy Mini column RNA extraction kit (Qiagen). After degrading the genomic DNA with RQ1 RNase-free DNase (Promega), cDNAs were synthesized by TaqMan reverse transcription reagents (Applied Biosystems) according to the manufacturer's instructions. The primer sequences have been described previously [1921] and are listed in Supplementary Table S1 (http://www.biochemj.org/bj/459/bj4590137add.htm). cDNAs and FastStart Universal SYBR Green Master Rox (Roche Applied Science) were applied to perform real-time PCR in a StepOne Plus system (Applied Biosystems).

RESULTS

Purification and identification of triglyceride accumulation factor from the silkworm haemolymph

Insects are one of the animal models used in studies of lipid metabolism and recent reports using Drosophila have revealed the conserved involvement of Hedgehog signalling in adipogenesis [22]. BmN4 cells, derived from the silkworm ovary, are proposed to be an insect triglyceride-accumulating cell differentiation model [19]. First, we found that silkworm haemolymph inhibited the proliferation of BmN4 cells (Figure 1A). After 3 days, BmFABP1 [B. mori fatty acid-binding protein 1; an aP2/Fabp4 (fatty acid-binding protein 4) homologue in silkworms] [19] gene expression and triglyceride storage had increased (Figures 1B–1D), which is similar to previously reported features of dexamethasone-induced triglyceride accumulation in cells [19].

Triglyceride accumulation in BmN4 cells induced by silkworm haemolymph

Figure 1
Triglyceride accumulation in BmN4 cells induced by silkworm haemolymph

(A) Inhibitory effects of the silkworm haemolymph on the growth of BmN4 cells. BmN4 cells were incubated in culture media containing 0% (●), 5% (▲) or 10% (■) silkworm haemolymph. Cell numbers were counted by a cytometer. (BD) Triglyceride accumulation phenotypes observed in BmN4 cells treated with silkworm haemolymph. After 3 days of incubation with or without 10% haemolymph, BmN4 cells were collected and subjected to either qPCR analysis for the BmFABP1 gene [normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA levels] (B), Oil Red O staining (C) or intracellular triglyceride quantification (D). Results are means±S.E.M. for three experiments. Statistical significance was determined by Student's t test (*P<0.05).

Figure 1
Triglyceride accumulation in BmN4 cells induced by silkworm haemolymph

(A) Inhibitory effects of the silkworm haemolymph on the growth of BmN4 cells. BmN4 cells were incubated in culture media containing 0% (●), 5% (▲) or 10% (■) silkworm haemolymph. Cell numbers were counted by a cytometer. (BD) Triglyceride accumulation phenotypes observed in BmN4 cells treated with silkworm haemolymph. After 3 days of incubation with or without 10% haemolymph, BmN4 cells were collected and subjected to either qPCR analysis for the BmFABP1 gene [normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA levels] (B), Oil Red O staining (C) or intracellular triglyceride quantification (D). Results are means±S.E.M. for three experiments. Statistical significance was determined by Student's t test (*P<0.05).

We then tried to purify the triglyceride accumulation factor in the silkworm haemolymph. Generally, terminally differentiated cells cease growing and acquire mature functions. Therefore we hypothesized that the triglyceride accumulation effect and the growth inhibitory activity are coupled. We evaluated the growth inhibitory activity of the haemolymph on the basis of [3H]thymidine incorporation into BmN4 cells. Silkworm haemolymph added to the BmN4 cell culture inhibited [3H]thymidine incorporation in a dose-dependent manner (Figure 2A). The inhibition of [3H]thymidine incorporation was decreased by pre-treatment of the haemolymph with proteinase (Figure 2B), indicating that a protein was responsible for the inhibitory activity. Purification was performed by ammonium sulfate precipitation and successive column chromatographies, as detailed in the Experimental section (Table 1). Gel filtration with the Superose 12 revealed a correlation between the protein measurement and the inhibition of [3H]thymidine incorporation into BmN4 cells (Figure 2C). SDS/PAGE analysis of the fractions showed a single 15-kDa protein band (Figure 2D). Analysis of amino acid sequences of two tryptic digests of the 15-kDa protein matched a part of a protein amino acid sequence that was previously reported as Bombyx mori PP [18] (Supplementary Figure S1A at http://www.biochemj.org/bj/459/bj4590137add.htm). This protein is an orthologue of the human NPC2 protein [11]. In the following sections, we refer to this protein as NPC2.

Table 1
Purification of a [3H]thymidine incorporation-inhibitory factor from silkworm haemolymph

Haemolymph (150 ml) was collected from 300 larvae and used as Fraction I. Detailed procedures of column chromatography are described in the Experimental section. One unit is defined as the amount of activity that inhibited 30% [3H]thymidine incorporation into BmN4 cells.

Fraction Total activity (units) Protein (mg) Specific activity (units/mg) Recovery (%) Purification (fold) 
I. Haemolymph 3500 2100 1.7 100 
II. Ammonium sulfate 2600 1600 1.6 74 0.9 
III. Heparin Toyopearl 660 58 11 19 6.5 
IV. Butyl Toyopearl 320 8.6 37 9.1 22 
V. Mono S 500 2.5 200 14 120 
VI. Superose 12 850 1.8 470 24 280 
Fraction Total activity (units) Protein (mg) Specific activity (units/mg) Recovery (%) Purification (fold) 
I. Haemolymph 3500 2100 1.7 100 
II. Ammonium sulfate 2600 1600 1.6 74 0.9 
III. Heparin Toyopearl 660 58 11 19 6.5 
IV. Butyl Toyopearl 320 8.6 37 9.1 22 
V. Mono S 500 2.5 200 14 120 
VI. Superose 12 850 1.8 470 24 280 

Purification of [3H]thymidine incorporation-inhibitory factor from silkworm haemolymph

Figure 2
Purification of [3H]thymidine incorporation-inhibitory factor from silkworm haemolymph

(A) Inhibitory effects of silkworm haemolymph on [3H]thymidine incorporation into BmN4 cells. BmN4 cells were incubated in culture medium containing 0, 1.3, 2.5, 5.0 or 10% haemolymph for 24 h. Cell cultures were then supplied with [3H]thymidine followed by 6 h incubation, and radioactivity of acid-insoluble fractions was measured with a liquid scintillation counter. (B) Effect of proteinase treatment on haemolymph-dependent inhibition of [3H]thymidine incorporation. BmN4 cells were incubated with 10% haemolymph pre-treated with proteinase K and pronase E, and [3H]thymidine incorporation was determined after 24 h. Each value was normalized to [3H]thymidine incorporation in cells incubated without haemolymph. Results are means±S.E.M. for four experiments. Statistical significance was determined by Student's t test (*P<0.05). (C) [3H]thymidine incorporation-inhibitory activities and protein amounts in fractions of Superose 12 gel filtration. (D) SDS/PAGE of Superose 12 fractions. Samples were applied to 15% gels and bands were detected by Coomassie Brilliant Blue staining. Numbers correspond to fractions shown in (C). M, molecular mass marker.

Figure 2
Purification of [3H]thymidine incorporation-inhibitory factor from silkworm haemolymph

(A) Inhibitory effects of silkworm haemolymph on [3H]thymidine incorporation into BmN4 cells. BmN4 cells were incubated in culture medium containing 0, 1.3, 2.5, 5.0 or 10% haemolymph for 24 h. Cell cultures were then supplied with [3H]thymidine followed by 6 h incubation, and radioactivity of acid-insoluble fractions was measured with a liquid scintillation counter. (B) Effect of proteinase treatment on haemolymph-dependent inhibition of [3H]thymidine incorporation. BmN4 cells were incubated with 10% haemolymph pre-treated with proteinase K and pronase E, and [3H]thymidine incorporation was determined after 24 h. Each value was normalized to [3H]thymidine incorporation in cells incubated without haemolymph. Results are means±S.E.M. for four experiments. Statistical significance was determined by Student's t test (*P<0.05). (C) [3H]thymidine incorporation-inhibitory activities and protein amounts in fractions of Superose 12 gel filtration. (D) SDS/PAGE of Superose 12 fractions. Samples were applied to 15% gels and bands were detected by Coomassie Brilliant Blue staining. Numbers correspond to fractions shown in (C). M, molecular mass marker.

We produced recombinant NPC2 by a baculovirus expression system (Supplementary Figure S1B). The affinity-purified fraction exhibited inhibitory activity on [3H]thymidine incorporation into BmN4 cells. The specific activity of the recombinant NPC2 was approximately half that of the fraction purified from silkworm haemolymph (Table 2). These findings, taken together, indicate that a protein responsible for the inhibition of [3H]thymidine incorporation into BmN4 cells was purified and that the protein was NPC2. We further demonstrated that recombinant NPC2 inhibited the proliferation of BmN4 cells (Figure 3A). BmFABP1 mRNA levels were increased after 2 days of culture (Figure 3B). In addition, lipid droplets stained with Oil Red O formed in BmN4 cells (Figure 3C). There was also an increased amount of triglyceride in the BmN4 cells (Figure 3D). Together, these findings indicated that NPC2 induced triglyceride accumulation in BmN4 cells.

Table 2
Specific activity of purified recombinant NPC2

[3H]thymidine incorporation-inhibitory activities of fractions purified from either silkworm haemolymph (Fraction VI) or the Sf-9 culture supernatant were determined. WT, wild-type.

Fraction Specific activity (units/mg) 
VI. Superose 12 450 
Recombinant WT 260 
Recombinant F90A 250 
Fraction Specific activity (units/mg) 
VI. Superose 12 450 
Recombinant WT 260 
Recombinant F90A 250 

Triglyceride accumulation in BmN4 cells induced by NPC2

Figure 3
Triglyceride accumulation in BmN4 cells induced by NPC2

(A) Inhibitory effects of NPC2 on BmN4 cell growth. BmN4 cells were incubated in culture medium containing the recombinant NPC2. Cell numbers on the second day of incubation were counted by a cytometer. (BD) Triglyceride-accumulating cell phenotypes observed in BmN4 cells treated with NPC2. On the second day of incubation with recombinant NPC2, BmN4 cells were collected and subjected to either qPCR analysis for the BmFABP1 gene [normalized to Rpl (ribosomal protein S4) mRNA levels] (B), Oil Red O staining (C) or intracellular triglyceride quantification (D). Results are means±S.E.M. for three or four experiments.

Figure 3
Triglyceride accumulation in BmN4 cells induced by NPC2

(A) Inhibitory effects of NPC2 on BmN4 cell growth. BmN4 cells were incubated in culture medium containing the recombinant NPC2. Cell numbers on the second day of incubation were counted by a cytometer. (BD) Triglyceride-accumulating cell phenotypes observed in BmN4 cells treated with NPC2. On the second day of incubation with recombinant NPC2, BmN4 cells were collected and subjected to either qPCR analysis for the BmFABP1 gene [normalized to Rpl (ribosomal protein S4) mRNA levels] (B), Oil Red O staining (C) or intracellular triglyceride quantification (D). Results are means±S.E.M. for three or four experiments.

Induction of triglyceride accumulation in the fat body tissues of living silkworms

Next, we examined whether NPC2 induced triglyceride accumulation in living silkworm larvae. We injected NPC2 into the silkworm haemolymph and measured BmFABP1 mRNA and triglyceride levels in the fat body. The findings indicate that both BmFABP1 mRNA and triglyceride levels in the fat body were increased by NPC2 (Figures 4A and 4B).

To elucidate the physiological relevance of NPC2, we administered antiserum to deplete the endogenous protein in living silkworms and calculated the triglyceride/DNA ratios in the fat body tissues. Injection of antiserum decreased triglyceride levels approximately 20% compared with injection of normal rabbit serum (Figure 4C). These findings suggest that NPC2 induces triglyceride accumulation in living silkworms.

In vivo up-regulation of triglyceride accumulation in silkworm fat body tissues by NPC2

Figure 4
In vivo up-regulation of triglyceride accumulation in silkworm fat body tissues by NPC2

(A) Up-regulation of BmFABP1 gene expression in silkworm fat body tissues induced by NPC2. Silkworm larvae (day 2 of 5th instar; 1.9±0.2 g/larva) were injected with 50 μl of buffer or the recombinant NPC2 (300 μg/ml). After 72 h, RNAs were extracted from the fat body. The amount of BmFABP1 gene mRNA was analysed by qPCR and normalized to that of an internal control gene, Rpl. (B) Stimulation of triglyceride accumulation in silkworm fat body tissues induced by NPC2. Silkworm larvae were injected with the recombinant NPC2 as described above. After 24 h, fat body tissues were dissected and lysates prepared. Total lipid fractions were obtained and the amounts of accumulated triglyceride were measured by the GPO-DAOS method. DNA quantities were determined from A280 values in water-dissolved ethanol precipitates of DNA extracted from tissues by the PCI method. The triglyceride and DNA ratios were calculated and values relative to non-injected groups are shown on the y-axis. Results are means±S.E.M. for three or four experiments. (C) Reduction of triglyceride accumulation by injection of antiserum. Silkworm larvae were injected with 50 μl of anti-silkworm NPC2 antiserum or normal rabbit serum. After 72 h, relative triglyceride levels were determined using the method described above. Results are means±S.E.M. for eight experiments. Statistical significance was determined by Student's t test (*P<0.05).

Figure 4
In vivo up-regulation of triglyceride accumulation in silkworm fat body tissues by NPC2

(A) Up-regulation of BmFABP1 gene expression in silkworm fat body tissues induced by NPC2. Silkworm larvae (day 2 of 5th instar; 1.9±0.2 g/larva) were injected with 50 μl of buffer or the recombinant NPC2 (300 μg/ml). After 72 h, RNAs were extracted from the fat body. The amount of BmFABP1 gene mRNA was analysed by qPCR and normalized to that of an internal control gene, Rpl. (B) Stimulation of triglyceride accumulation in silkworm fat body tissues induced by NPC2. Silkworm larvae were injected with the recombinant NPC2 as described above. After 24 h, fat body tissues were dissected and lysates prepared. Total lipid fractions were obtained and the amounts of accumulated triglyceride were measured by the GPO-DAOS method. DNA quantities were determined from A280 values in water-dissolved ethanol precipitates of DNA extracted from tissues by the PCI method. The triglyceride and DNA ratios were calculated and values relative to non-injected groups are shown on the y-axis. Results are means±S.E.M. for three or four experiments. (C) Reduction of triglyceride accumulation by injection of antiserum. Silkworm larvae were injected with 50 μl of anti-silkworm NPC2 antiserum or normal rabbit serum. After 72 h, relative triglyceride levels were determined using the method described above. Results are means±S.E.M. for eight experiments. Statistical significance was determined by Student's t test (*P<0.05).

Molecular mechanisms of triglyceride accumulation in BmN4 cells by NPC2

Mammalian NPC2 protein binds cholesterol and regulates cellular cholesterol levels [5,13]. Some reports also indicate that NPC2 protein controls haemapoietic cell differentiation and that this activity is independent of its cholesterol-binding capacity [8]. NPC2 protein is incorporated into cells through binding to mannose 6-phosphate receptors, and the addition of mannose 6-phosphate to the culture medium competitively inhibits this process [5]. Both the cholesterol binding-dependent and -independent functions of NPC2 protein are mediated by a mannose 6-phosphate-inhibitable pathway [8,13]. We found that the [3H]thymidine incorporation-inhibitory activity of NPC2 was sensitive to mannose 6-phosphate (Figure 5A). Moreover, NPC2-dependent triglyceride accumulation in BmN4 cells was also attenuated by mannose 6-phosphate (Figure 5B). To test whether NPC2 was incorporated into BmN4 cells, we prepared BmN4 cell lysate after a 24-h incubation in the presence of NPC2. NPC2 was detected in the lysate of cells supplied with the recombinant protein, whereas the band intensity was reduced by mannose 6-phosphate (Figure 5C, and Supplementary Figure S2 at http://www.biochemj.org/bj/459/bj4590137add.htm). Taken together, these results suggest that NPC2 was incorporated into BmN4 cells via binding to mannose 6-phosphate receptors, and that this incorporation process was required for the triglyceride accumulation induced by NPC2. To further elucidate the mode of action, we constructed a mutant NPC2 in which residue 90 (phenylalanine), which is required for cholesterol-binding of mammalian NPC2 protein [13], was substituted with alanine (Figure 6A, and Supplementary Figure S3 at http://www.biochemj.org/bj/459/bj4590137add.htm). The mutated NPC2 (F90A) completely lost its cholesterol-binding capacity (Figure 6B), whereas its [3H]thymidine incorporation-inhibitory activity was comparable with that of the wild-type protein (Table 2). The F90A mutant also showed promotive effects on BmFABP1 gene expression and lipid accumulation in BmN4 cells (Figures 6C and 6D), indicating that the triglyceride accumulation activity of NPC2 is independent of the cholesterol-binding capacity.

Effects of mannose 6-phosphate on triglyceride accumulation in BmN4 cells induced by NPC2

Figure 5
Effects of mannose 6-phosphate on triglyceride accumulation in BmN4 cells induced by NPC2

(A) BmN4 cells were incubated with the recombinant NPC2 (10 μg/ml) and in the presence or absence of 25 mM mannose 6-phosphate (M6P). After 24 h, [3H]thymidine incorporation was measured, and values relative to those of cells incubated without NPC2 are shown on the y-axis. (B) After 48 h, triglyceride accumulated in BmN4 cells was quantified. Results are means±S.E.M. for three or four experiments. Statistical significance was determined by Student's t test (*P<0.05). (C) Incorporation of extracellular NPC2 into BmN4 cells. BmN4 cells were incubated in the presence of recombinant NPC2 with or without 25 mM mannose 6-phosphate for 24 h. Total cell lysate was subjected to immunoblot analysis with anti-NPC2 antiserum to detect incorporated proteins.

Figure 5
Effects of mannose 6-phosphate on triglyceride accumulation in BmN4 cells induced by NPC2

(A) BmN4 cells were incubated with the recombinant NPC2 (10 μg/ml) and in the presence or absence of 25 mM mannose 6-phosphate (M6P). After 24 h, [3H]thymidine incorporation was measured, and values relative to those of cells incubated without NPC2 are shown on the y-axis. (B) After 48 h, triglyceride accumulated in BmN4 cells was quantified. Results are means±S.E.M. for three or four experiments. Statistical significance was determined by Student's t test (*P<0.05). (C) Incorporation of extracellular NPC2 into BmN4 cells. BmN4 cells were incubated in the presence of recombinant NPC2 with or without 25 mM mannose 6-phosphate for 24 h. Total cell lysate was subjected to immunoblot analysis with anti-NPC2 antiserum to detect incorporated proteins.

Triglyceride accumulation in BmN4 cells induced by cholesterol-binding-deficient NPC2

Figure 6
Triglyceride accumulation in BmN4 cells induced by cholesterol-binding-deficient NPC2

(A) Alignment of amino acid sequences of silkworm and human NPC2 protein. The boxed residues indicates residue 90 (phenylalanine). (B) The cholesterol-binding assay. Each recombinant protein (50 μg) was incubated in a labelling solution supplied with different concentrations of [3H]cholesterol. Free or bound cholesterols were separated by centrifugation through a CentriSep gel-filtration column. Radioactivity of passed-through fractions was measured with a liquid scintillation counter. (C and D) BmN4 cells were incubated in culture medium containing NPC2 with the F90A mutation. On the second day of incubation, the cells were collected and subjected to either qPCR analysis for the BmFABP1 gene (normalized to Rpl mRNA levels) (C) or intracellular triglyceride quantification (D). Results are means±S.E.M. for three experiments. (C) Fold changes relative to levels without NPC2 are shown.

Figure 6
Triglyceride accumulation in BmN4 cells induced by cholesterol-binding-deficient NPC2

(A) Alignment of amino acid sequences of silkworm and human NPC2 protein. The boxed residues indicates residue 90 (phenylalanine). (B) The cholesterol-binding assay. Each recombinant protein (50 μg) was incubated in a labelling solution supplied with different concentrations of [3H]cholesterol. Free or bound cholesterols were separated by centrifugation through a CentriSep gel-filtration column. Radioactivity of passed-through fractions was measured with a liquid scintillation counter. (C and D) BmN4 cells were incubated in culture medium containing NPC2 with the F90A mutation. On the second day of incubation, the cells were collected and subjected to either qPCR analysis for the BmFABP1 gene (normalized to Rpl mRNA levels) (C) or intracellular triglyceride quantification (D). Results are means±S.E.M. for three experiments. (C) Fold changes relative to levels without NPC2 are shown.

The responsible intracellular pathways acting downstream of extracellular NPC2 remain to be elucidated. We focused on the phosphorylation cascade involved in both cell growth and triglyceride accumulation. Extracellular NPC2 activated AMPK and inactivated ERK in BmN4 cells (Figure 7A). An AMPK inhibitor, compound C, blocked the triglyceride accumulation activity of NPC2 (Figure 7B). These findings suggest that NPC2 induced triglyceride accumulation via AMPK activation.

Analysis of phosphorylation cascades regulated by extracellular NPC2

Figure 7
Analysis of phosphorylation cascades regulated by extracellular NPC2

(A) Activation of AMPK and inactivation of ERK by NPC2. BmN4 cells were incubated with or without 20 μg/ml NPC2. After 24 h, the cells were washed and lysed. Cell lysates were separated by SDS/PAGE, and AMPK, ERK and β-actin were detected by immunoblotting. (B) Effects of an AMPK inhibitor on triglyceride accumulation in BmN4 cells induced by NPC2. BmN4 cells were incubated with the recombinant NPC2 (10 μg/ml) in the presence or absence of 5 μM compound C. After 48 h, the triglyceride accumulation in BmN4 cells was quantified. Results are means±S.E.M. for three experiments. Statistical significance was determined by Student's t test (*P<0.05).

Figure 7
Analysis of phosphorylation cascades regulated by extracellular NPC2

(A) Activation of AMPK and inactivation of ERK by NPC2. BmN4 cells were incubated with or without 20 μg/ml NPC2. After 24 h, the cells were washed and lysed. Cell lysates were separated by SDS/PAGE, and AMPK, ERK and β-actin were detected by immunoblotting. (B) Effects of an AMPK inhibitor on triglyceride accumulation in BmN4 cells induced by NPC2. BmN4 cells were incubated with the recombinant NPC2 (10 μg/ml) in the presence or absence of 5 μM compound C. After 48 h, the triglyceride accumulation in BmN4 cells was quantified. Results are means±S.E.M. for three experiments. Statistical significance was determined by Student's t test (*P<0.05).

The effect of NPC2 proteins on mammalian cell lines

3T3-L1 fibroblasts are widely used to study mammalian adipocyte differentiation and physiology [23]. To investigate whether the adipogenic properties of NPC2 proteins are conserved in mammals, we produced recombinant murine NPC2 protein (Supplementary Figure S4 at http://www.biochemj.org/bj/459/bj4590137add.htm) and tested its effects on 3T3-L1 cells. Murine NPC2 protein up-regulated the mRNA level of aP2/Fabp4, a mammalian adipocyte differentiation marker gene (Figure 8A). This promotion was dependent on the presence of both dexamethasone and IBMX (results not shown). Amino acid sequences of NPC2 protein and its cholesterol-binding capacity are highly conserved in eukaryotes. Because yeast Npc2p is capable of replacing the functions of human NPC2 in human fibroblasts [11], we hypothesized that silkworm NPC2 and murine NPC2 are interchangeable. Silkworm NPC2 induced a relatively weak up-regulation of aP2/Fabp4 gene expression in 3T3-L1 cells (Figure 8B). Similarly, murine NPC2 protein promoted BmFABP1 gene expression in BmN4 cells (Figure 8C). These findings suggest that the functions of NPC2 protein in terms of adipose tissue-related gene expression are conserved.

Enhancement of adipose tissue-related gene expression by insect and mammalian NPC2 proteins

Figure 8
Enhancement of adipose tissue-related gene expression by insect and mammalian NPC2 proteins

(A and B) 3T3-L1 cells were cultured in DMEM supplied with 10% calf serum at 37°C with 5% CO2. Adipocyte differentiation of 3T3-L1 cells was induced by culturing cells in medium containing 1 μM dexamethasone and 0.5 mM IBMX. During this differentiation process, we assessed the effects of supplementation with insulin (1 μg/ml), murine NPC2 protein (20 μg/ml) (A) or silkworm NPC2 (20 μg/ml) (B). After a 3 day incubation, the cells were collected and subjected to qPCR analysis for the aP2/Fabp4 gene [normalized to arbp (acidic ribosomal phosphoprotein) mRNA levels]. (C) Promotive effect of murine NPC2 protein on adipocyte differentiation in BmN4 cells. On day 2 of incubation with recombinant murine NPC2 protein, BmN4 cells were collected and subjected to qPCR analysis for the BmFABP1 gene (normalized to Rpl mRNA levels). Fold-changes relative to cells without supplementation are shown. Results are means±S.E.M. for three experiments. Statistical significance compared with each non-treated group was determined by Student's t test (*P<0.05).

Figure 8
Enhancement of adipose tissue-related gene expression by insect and mammalian NPC2 proteins

(A and B) 3T3-L1 cells were cultured in DMEM supplied with 10% calf serum at 37°C with 5% CO2. Adipocyte differentiation of 3T3-L1 cells was induced by culturing cells in medium containing 1 μM dexamethasone and 0.5 mM IBMX. During this differentiation process, we assessed the effects of supplementation with insulin (1 μg/ml), murine NPC2 protein (20 μg/ml) (A) or silkworm NPC2 (20 μg/ml) (B). After a 3 day incubation, the cells were collected and subjected to qPCR analysis for the aP2/Fabp4 gene [normalized to arbp (acidic ribosomal phosphoprotein) mRNA levels]. (C) Promotive effect of murine NPC2 protein on adipocyte differentiation in BmN4 cells. On day 2 of incubation with recombinant murine NPC2 protein, BmN4 cells were collected and subjected to qPCR analysis for the BmFABP1 gene (normalized to Rpl mRNA levels). Fold-changes relative to cells without supplementation are shown. Results are means±S.E.M. for three experiments. Statistical significance compared with each non-treated group was determined by Student's t test (*P<0.05).

In human adipocytes, knockdown of the NPC2 gene impairs triglyceride accumulation via the up-regulation of lipolysis- and β oxidation-related genes [14]. We therefore considered that supplementation with murine NPC2 protein altered the levels of triglyceride accumulation as well as the expression of these lipid metabolism genes. As observed in BmN4 cells treated with silkworm NPC2 (Figure 3D), we confirmed that murine NPC2 protein also promoted the accumulation of triglyceride in 3T3-L1 cells (Figure 9A). Moreover, the addition of NPC2 protein to the culture medium led to a decrease in the mRNA levels of several lipolysis- and β oxidation-related genes, such as Pnpla2 (adipocyte triglyceride lipase), Cpt1b (carnitine palmitoyl transferase) and Acadm (acyl-coenzyme A dehydrogenase medium chain) (Figure 9B). The opposite effects after the addition of NPC2 demonstrated above appear to be consistent with the effects of the NPC2 gene knockdown reported previously [14]. These findings suggest that NPC2 targets these lipid metabolism genes and therefore affects lipolysis and β oxidation, leading to triglyceride accumulation in 3T3-L1 cells.

Effects of murine NPC2 protein on triglyceride accumulation and expression of lipid metabolism genes in 3T3-L1 adipocytes

Figure 9
Effects of murine NPC2 protein on triglyceride accumulation and expression of lipid metabolism genes in 3T3-L1 adipocytes

(A) 3T3-L1 adipocytes were pre-cultured in serum-free medium for 24 h before treatment. Cells were incubated in the presence of 20 μg/ml murine NPC2 protein for another 24 h. The cells were then collected using trypsin/EDTA and lysed for lipid extraction, as described above. Triglyceride amounts relative to the control group were determined. Results are means±S.E.M. for two experiments (*P<0.05). (B) 3T3-L1 adipocytes treated with 20 μg/ml murine NPC2 protein were collected and subjected to qPCR analysis for Pnpla2, Cpt1b and Acadm (normalized to arbp mRNA levels). Fold-changes relative to cells without added NPC2 are shown. Results are means±S.E.M. for three experiments. Statistical significance compared with each non-treated group was determined by Student's t test (*P<0.05).

Figure 9
Effects of murine NPC2 protein on triglyceride accumulation and expression of lipid metabolism genes in 3T3-L1 adipocytes

(A) 3T3-L1 adipocytes were pre-cultured in serum-free medium for 24 h before treatment. Cells were incubated in the presence of 20 μg/ml murine NPC2 protein for another 24 h. The cells were then collected using trypsin/EDTA and lysed for lipid extraction, as described above. Triglyceride amounts relative to the control group were determined. Results are means±S.E.M. for two experiments (*P<0.05). (B) 3T3-L1 adipocytes treated with 20 μg/ml murine NPC2 protein were collected and subjected to qPCR analysis for Pnpla2, Cpt1b and Acadm (normalized to arbp mRNA levels). Fold-changes relative to cells without added NPC2 are shown. Results are means±S.E.M. for three experiments. Statistical significance compared with each non-treated group was determined by Student's t test (*P<0.05).

DISCUSSION

We have previously reported that silkworms are advantageous for biochemical and pharmaceutical experiments because of their large body size [2428]. In the present study, we aimed to identify a humoral factor that regulates triglyceride accumulation. We purified and identified secreted NPC2 protein as a triglyceride accumulation factor in insects. Induction of triglyceride accumulation by NPC2 protein was independent of its cholesterol-binding capacity and dependent on the AMPK pathway.

A previous study demonstrated that knockdown of the NPC2 gene in mature adipocytes suppresses triglyceride storage [14] and therefore the NPC2 gene is assumed to be required for maintaining the function of mature adipocytes. To our knowledge, however, there are no studies showing that extracellular NPC2 protein promotes triglyceride accumulation.

In mammals, both the cholesterol binding-dependent and -independent activities of the NPC2 protein are suppressed by mannose 6-phosphate, suggesting that mannose 6-phosphate receptors mediate NPC2 activity [5,29]. We also showed in BmN4, an insect cell line, that silkworm NPC2 was detected in the cellular fraction after incubation and the addition of mannose 6-phosphate decreased the NPC2 protein levels. This finding indicates that extracellular NPC2 protein shares the mannose 6-phosphate receptor pathway in both insects and mammals. In previous reports, it was assumed that NPC2 protein was internalized via mannose 6-phosphate receptors for transporting intracellular cholesterol [5,29]. Future studies are required to reveal factors (other receptors or signalling molecules) connecting NPC2 internalization and AMPK activation, leading to triglyceride accumulation.

NPC2 protein is a cholesterol-binding protein. In fibroblasts derived from human patients with NPC2, an unusual accumulation of LDL (low-density-lipoprotein)-derived cholesterol in endolysosomal compartments is observed. This cholesterol accumulation is reverted by the addition of NPC2 protein to the culture medium [5]. The rescue effect of NPC2 protein requires its cholesterol-binding capacity. Therefore NPC2 protein is involved in cholesterol homoeostasis as a cholesterol carrier. A previous study indicated that NPC2 has cholesterol-binding-independent activities [8]. We showed that cholesterol-binding capacity was dispensable for NPC2-dependent triglyceride accumulation. This finding provides us more information about the cholesterol binding-independent activities of the NPC2 protein.

The responsible intracellular pathways acting downstream of extracellular NPC2 protein are poorly understood. A previous report showed that knockout of NPC2 in fibroblasts results in the activation of ERK, a MAPK (mitogen-activated protein kinase) involved in cell growth [30]. We found that extracellular NPC2 inhibited ERK activation in BmN4 cells. It is reasonable to consider that ERK inactivation promotes triglyceride storage, because ERK phosphorylates hormone-sensitive lipase [31,32]. Therefore ERK inactivation by the NPC2 protein might be involved in both the cessation of cell growth and the enhancement of triglyceride accumulation. We further elucidated that AMPK was one of the kinases responsible for NPC2 protein-dependent triglyceride accumulation. The roles of AMPK in lipid metabolism have been studied in various cell types and tissues. Activated AMPK induces lipid oxidation in the skeletal muscle and liver [33]. On the other hand, apparently different actions of AMPK are also reported in adipocytes where activation of AMPK has anti-lipolytic effects [34,35]. Furthermore, on the basis of previous studies demonstrating that adiponectin, a potent activator of AMPK, promotes adipocyte differentiation [16], we assumed the possible involvement of AMPK in NPC2-dependent lipid accumulation and further tested the hypothesis. In the present study, we showed that extracellular NPC2 protein activates AMPK, and an AMPK inhibitor abolishes NPC2 activity. This is the first example of NPC2 activating AMPK. These results provide new insight into the biochemical activity of NPC2 protein as an extracellular signalling molecule.

NPC2 protein may play a crucial role in cellular differentiation and development. The addition of silkworm haemolymph or purified NPC2 to BmN4 cell cultures induced growth inhibition, lipid droplet formation and up-regulation of BmFABP1 (an aP2/Fabp4 homologue in silkworms [19]) gene expression. Holometabola insects that undergo complete metamorphosis, such as silkworms, accumulate a large amount of fat during the larval stages. The fat may be utilized as an essential energy reservoir for the upcoming food deprivation period during the pupal stages. Because NPC2 induced triglyceride accumulation in the fat body tissues of silkworm larvae, we speculate that it has a facilitative role in lipid accumulation at the larval stages. BmN4 cells are proposed to be a model cell line for lipid-accumulating cells that are major constituents of the insect fat body tissue. Our findings regarding triglyceride accumulation in BmN4 cells might be applicable to triglyceride accumulation in mammalian white adipocytes, a cell type responsible for lipid accumulation in vertebrates. The above effects of NPC2 on silkworm cells are consistent with characteristic features of adipocyte differentiation of 3T3-L1 cells. Furthermore, recent studies revealed that mammalian NPC2 is involved in blood cell differentiation [8]. Therefore we assumed that secreted NPC2 protein has the potential to control cellular differentiation. We observed NPC2-dependent increases in the mRNA levels of marker genes (Figure 8A) and triglyceride accumulation (Figure 9A) in 3T3-L1 cells under conditions without insulin supplementation. Therefore NPC2 functions seem not to require adipocytes in a fully differentiated state. In addition, we found that NPC2 increased the triglyceride content in CHO-K1 cells, a non-adipocyte cell line (Supplementary Figure S5 at http://www.biochemj.org/bj/459/bj4590137add.htm), suggesting that the effect of NPC2 on cellular triglyceride contents is not limited to adipocytes. On the basis of these results, we consider that NPC2 might exert modulatory effects on various cell types and thus has the potential to impact basic physiological aspects. Further studies are needed to elucidate the role of this protein in the cellular differentiation and development of insects and other animals.

Abbreviations

     
  • Acadm

    acyl-coenzyme A dehydrogenase medium chain

  •  
  • AMPK

    AMP-activated protein kinase

  •  
  • arbp

    acidic ribosomal phosphoprotein

  •  
  • BmFABP1

    B. mori fatty acid-binding protein 1

  •  
  • Cpt1b

    carnitine palmitoyl transferase

  •  
  • DAOS

    N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt

  •  
  • DMEM

    Dulbecco’s modified Eagle’s medium

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • Fabp4

    fatty acid-binding protein 4

  •  
  • GPO

    glycerol-3-phosphate oxidase

  •  
  • IBMX

    isobutylmethylxanthine

  •  
  • PCI

    phenol-chloroform-isoamyl alcohol

  •  
  • Pnpla2

    adipocyte triglyceride lipase

  •  
  • Pp

    promoting protein

  •  
  • NPC

    Niemann–Pick disease type C

  •  
  • qPCR

    quantitative real-time PCR

  •  
  • Rpl

    ribosomal protein S4

  •  
  • TCA

    trichloroacetic acid

AUTHOR CONTRIBUTION

Tatsuo Adachi conceived and designed the research, performed all of the experiments, analysed the data and drafted the paper. Kenichi Ishii, Yasuhiko Matsumoto, Yohei Hayashi and Hiroshi Hamamoto contributed important conceptual advice and critical revision of the paper. Kazuhisa Sekimizu directed the research and wrote the paper.

We thank Dr Makoto Arita (University of Tokyo, Tokyo, Japan) for providing the CHO-K1 cells.

FUNDING

This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science [grant number 24-10784].

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Supplementary data