Histology and molecular biology studies on the expression and localization of angiopoietin‑like protein 8 in human tissues
- Authors:
- Published online on: September 25, 2019 https://doi.org/10.3892/br.2019.1243
- Pages: 215-221
Abstract
Introduction
Angiopoietin-like protein (ANGPTL) 8 is a member of the ANGPTL family (1) and is also known as RIFL (2), TD26(3), lipasin (4), PRO1185, PVPA599, C19orf80 and betatrophin (5). ANGPTL8 is involved in the partitioning of triglycerides to muscle and adipose tissues in conjunction with ANGPTL3 and ANGPTL4 (6,7). They regulate the activity of lipoprotein lipase (LPL) in organs (7). ANGPTL8 interacts with ANGPTL3 and inhibits LPL in muscle tissue, whereas ANGPTL4 inhibits LPL in adipose tissue.
The ANGPTL8 gene is located on chromosome 19 and consists of four exons that encode 198 amino acids. The protein contains a signal peptide consisting of 20 amino acids at the N-terminus, the specific epitope 1 domain and two coiled coil domains (Fig. 1A). Specific epitope 1 domain is necessary for inhibition of LPL, and two coiled coil domains are necessary for the complex formation with ANGPTL3 and ANGPTL4 (6,7). The protein is processed to the mature protein by cleavage of signal peptide and released into the bloodstream (1).
It was reported that ANGPTL8 is highly expressed in the liver, white and brown adipose tissue (BAT) (2,5,6). In vitro studies demonstrated that the expression level of ANGPTL8 is altered depending on the differentiation and functional state of the cells (2). However, the precise localization and distribution of ANGPTL8-expressing cells in these organs remains unclear. The elucidation of the localization of ANGPTL8 in these organs will contribute to the understanding of the physiological roles of ANGPTL8 and the association with local lipid metabolism. Further, this may aid the understanding of the pathological role of ANGPTL8 in metabolic diseases and the development of novel therapies for these diseases.
The aim of the present study was to investigate the expression and localization of ANGPTL8 in normal human tissues. Using formalin-fixed paraffin-embedded (FFPE) specimens, ANGPTL8 expression levels and localization were examined by molecular biological methods. During the cloning of ANGPTL8 mRNA from normal liver, splice variants were identified. The structures of the splice variants were also documented.
Materials and methods
Sequencing of ANGPTL8 mRNA from the liver
The coding sequence of ANGPTL8 was amplified and cloned from the total RNA of the liver (cat. no. K4000-1; lot no. 0111320; Thermo Fisher Scientific, Inc.). Total RNA (2 µg) was treated with DNase I (Thermo Fisher Scientific, Inc.) at room temperature (RT) for 15 min, and cDNA was synthesized by random primer method using the SuperScript III First Strand Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. PCR was performed with cDNA transcribed from 100 ng total RNA using the AmpliTaq Gold PCR kit (Thermo Fisher Scientific, Inc.). The primers used were: Forward (F), 5'-TAGGCGCCCCCATGGGCGGCCCAGA-3' and reverse (R), 5'-GGCTGGGAGCGCCGCTGTGT-3' (Fig. 1A). The thermocycling conditions were as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for 1 min, 55˚C for 1 min and 72˚C for 1 min. The amplified PCR product was cloned into pCR-II TOPO (Thermo Fisher Scientific, Inc.) using the TA-cloning method. Cloned ANGPTL8 fragments were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific, Inc.).
Human tissue samples and histological assessment
FFPE specimens of lipoma, hibernoma and normal human tissues of the liver, adipose tissue, heart, stomach, small intestine, large intestine, pancreas, spleen, kidney, lymph node, bone marrow, thyroid, adrenal gland, ovary and testis were used in the present study. The normal human tissues were collected adjacent to malignant tumor tissues in resected organs. Lipomas and hibernomas were resected tumor tissues. The tissues were fixed in 10% formalin at RT for 24 h and embedded in paraffin. Three specimens of each normal tissue, lipoma and hibernoma were used for the study. Tissues were obtained from the archives of pathological specimens of Nippon Medical School Hospital (Tokyo, Japan) and were originally obtained between January 2014 and December 2018. The tissues obtained from 45 cases; median age, 65 (25-81) years. A total of 21 male and 24 female patients were recruited. The personal data were anonymized and only pathological diagnoses were available for the study. This study was performed in accordance with the principles of the Declaration of Helsinki, 2013 and the Japanese Society of Pathology, Ethics Committee. The study was approved by Ethics Committee of Nippon Medical School Hospital (approval no. 30-11-1304). Written informed consent was obtained from all patients at the time of hospitalization.
FFPE specimens of lipoma, hibernoma and normal human tissues were stained with hematoxylin and eosin. Briefly, 4-µm-thick sections were stained with hematoxylin for 5 min and eosin for 3 min at room temperature. The stained sections were observed using a light microscope (magnifications, x100, x200 and x400).
RNA extraction
Total RNA was extracted from FFPE specimens using the RNeasy FFPE kit (Qiagen, Inc.) according to the manufacturer's instructions. Briefly, five paraffin sections (4 µm) of a FFPE specimen were deparaffinized in 1.5-ml tubes. After the sections were dried, they were digested in PKD buffer with proteinase K at 56˚C until the tissues were completely dissolved. Total RNA was extracted, and the concentration was measured.
Reverse transcription-quantitative (RT-q)PCR of wild type ANGPTL8
The wild type ANGPTL8 mRNA (KF809856) was quantitated in the normal human tissues, lipoma and hibernoma. Total RNA (200 ng) were extracted from FFPE specimens and were treated with DNase I (Thermo Fisher Scientific, Inc.) at RT for 15 min. cDNA was synthesized using the SuperScript VILO cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The primers for wild type ANGPTL8 mRNA were: F1, 5'-CAGGAACAGCCTGGGTCTCTA-3' and R1, 5'-AGCTGCAGAATATCCTCCTCCAT-3' (Fig. 1A). For standardization, the expression level of 18S rRNA was quantified using the following primers: Forward, 5'-TAGCCTTTGCCATCACTGCC-3' and reverse, 5'-CACCACAACTCCTTTCGTCTGTAA-3'. The qPCR was performed in 20 µl reaction solution containing 1X PowerUp SYBR Green Master mix (Thermo Fisher Scientific, Inc.), 500 nM forward and reverse primers and cDNA transcribed from 100 ng total RNA as the template. The reaction was initiated at 50˚C for 2 min and 95˚C for 2 min, followed by 40 cycles of sequential incubations at 95˚C for 3 sec and 60˚C for 30 sec. The changes in fluorescence were monitored using a StepOne Plus Real-Time PCR system (Thermo Fisher Scientific, Inc.) and quantitation cycles (Cq) were determined. The expression level of wild type ANGPTL8 in the tissues was calculated as follows: ΔCq = Cq(wild type)-Cq(18S rRNA); and ΔΔCq = ΔCq(tissue)-ΔCq(liver). Expression levels were calculated following the 2-ΔΔCq method (8).
Expression ratio of splice variant 2/wild type ANGPTL8
The splice variant 2 of ANGPTL8 (KF809858) was quantified in liver, adipose tissue and hibernoma. The splice variant 2 was quantified using F1 and R2 primers (R2, 5'-TGTGGCTCTGCTTGTCA-3'; Fig. 1A). RT-qPCR was performed as detailed in the previous section. The expression ratio of splice variant 2/wild type ANGPTL8 was determined as follows: ΔCq = Cq(splice variant 2)-Cq(wild type). The expression ratio was obtained using the 2-ΔCq.
Immunohistochemistry (IHC)
IHC was performed on FFPE sections of normal human tissues, lipoma and hibernoma. Paraffin sections (4 µm) were deparaffinized in xylene and ethanol and hydrated in PBS. Sections were then treated with 10 mM citrate buffer (pH 6.0) at 121˚C for 15 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol at RT for 30 min. After incubation with 10% normal goat serum (Nichirei Biosciences, Inc.) at RT for 30 min, rabbit anti-ANGPTL8 antibody (cat. no. 7619; 1:500; ProSci, Inc.) was applied on the sections. In the negative control without an antibody, PBS was applied to the sections. Sections were incubated at 4˚C overnight. Subsequently, slides were incubated with a peroxidase-labeled anti-rabbit immunoglobulin antibody using the Histofine Simple Stain MAX-PO® (cat. no. 424141; prediluted; Nichirei Biosciences, Inc.) at RT for 1 h. Peroxidase activity was detected by incubation with diaminobenzidine at RT for 2 min using Histofine DAB Substrate kit (Nichirei Biosciences, Inc.) and the sections were counterstained with hematoxylin at RT for 1 min. The immunostained sections were observed using a light microscope (magnification, x200 and x400).
In situ hybridization (ISH)
ISH was performed with an ISH Reagent kit (cat. no. SRK-02; GenoStaff, Co., Ltd.) using FFPE sections (4 µm) of normal human tissues, lipoma, and hibernoma. Briefly, sections were deparaffinized, rehydrated in PBS and subsequently incubated in 10% neutral buffered formalin at RT for 15 min and treated with 20 µg/ml proteinase K (cat. no. S3004; Agilent Technologies, Inc.) in Tris-HCl (pH 7.6) at 37˚C for 10 min. After incubation in 10% neutral buffered formalin at RT for 15 min, samples were incubated in 0.2 N HCl at RT for 10 min. G-Hybo hybridization solution (100 µl; provided with kit) mixed with a digoxigenin-labeled anti-sense or sense probe was applied to the sections and incubated at 80˚C for 10 min. The anti-sense and sense probes were synthesized from the wild type ANGPTL8 using the T7/SP6 Digoxigenin Labeling kit (Roche Diagnostics, K.K.) according to the manufacturer's instructions. Following the initial incubation, sections were incubated at 50˚C overnight and washed twice with 1X Wash Buffer (provided with kit) at 50˚C for 10 min. Following blocking with G-Block (provided with kit) at RT for 30 min, sections were further incubated with an alkaline phosphatase-labeled anti-digoxigenin antibody (anti-digoxigenin-AP, Fab fragment; cat. no. 11093274910; 1:1,000; Roche Diagnostics) and the enzymatic activity was detected using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP Stock solution; cat. no. 11681451001; Roche Diagnostics) according to the manufacturer's instructions. Two types of negative control were used in the present study. In the first type of negative control, the slides were treated with 100 µg/ml RNase A (Takara Bio, Inc.) in 1X standard saline citrate (150 mM NaCl/15 mM citrate; pH 7.0) at 37˚C for 1 h to remove all endogenous RNA, and in the second type of negative control, the slides were incubated with 100 µl G-Hybo without probes at 80˚C for 10 min. Slides were further processed in the same manner as described. The stained sections were observed using a light microscope (magnification, x200 and x400).
Statistical analysis
Data are expressed as the mean ± standard deviation of three samples. The statistical analysis was performed using R (version 3.6.1; https://www.r-project.org). The statistical analysis among the organs was conducted using Kruskal Wallis followed by Dunn's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
Sequencing of ANGPTL8 mRNA
Amplification and cloning of ANGPTL8 mRNA identified three different fragments in the liver (Fig. 1A). One was KF809856, the wild type mRNA, and KF809857 and KF809858 are two were alternatively spliced mRNAs. The first splice variant (Splice Variant 1; KF809857) lacked 146 base pairs of exon 1. This deletion caused a frame shift producing a truncated protein containing 63 amino acids due to a premature stop codon in exon 2. The second splice variant (Splice Variant 2; KF809858) lacked 162 base pairs, the entire exon 2, and generated a short version of ANGPTL8. The splice variants contained two single nucleotide polymorphisms (SNPs), rs1541922 and rs192460764 (registered in dbSNP; available at https://www.ncbi.nlm.nih.gov/snp/). The SNP rs192460764 caused the replacement of Arg to Trp (p.Arg172Trp). The wild type ANGPTL8 did not contain these SNPs and no further SNPs were identified.
RT-qPCR of wild type ANGPTL8
The expression level of wild type ANGPTL8 mRNA was determined in the FFPE tissue specimens. Results showed ANGPTL8 levels were highest in the liver, adipose tissue, lipoma and hibernoma (Fig. 1B; Table I); levels in the other tissues were low. The level of ANGPTL8 in the liver was significantly increased compared with the other samples (P<0.05), except the adipose tissue, lipoma and hibernoma where differences were not significant (P>0.05). The levels in the adipose tissue, lipoma and hibernoma were also higher than other tissues.
Expression ratio of splice variant 2/wild type ANGPTL8
The level of ANGPTL8 splice variant 2 mRNA, which generates a shorter form of ANGPTL8 due to partial lack of coiled-coiled domains, was quantified in liver, adipose tissue and hibernoma. The level of splice variant 1, which generates a truncated form of ANGPTL8 and lacks almost entire coiled-coil domains, was not quantitated in the present study. The expression level of the splice variant 2 was below the detection limit in the adipose tissue and hibernoma, and the variant was detected only in the liver. The expression level of the splice variant 2 in the four liver tissues was very low and the relative level was 0.0069±0.0024, compared with the wild type ANGPTL8.
IHC
In the liver, expression of ANGPTL8 was observed in periportal zone 1 of hepatic acinus (Fig. 2A and B). In high magnification, the cytoplasm in the hepatocytes showed a positive reaction (Fig. 2C). The negative control without the primary antibody showed no signal (Fig. 2D). In the adipose tissue, expression of ANGPTL8 was observed in immature adipocytes (Fig. 2E; arrows). The cytoplasmic rims of mature adipocytes in the adipose tissue and lipoma were also stained (Fig. 2E and F). In hibernoma, the foamy cytoplasm of tumor cells was strongly stained (Fig. 2G). The negative control without the primary antibody showed no positive staining (Fig. 2H). Adipose tissue in the bone marrow showed a positive staining (Fig. 2I). The positive staining was not observed in the negative control without primary antibody (Fig. 2J). Positive staining was not observed in the epithelial cells of the small intestine (Fig. 2K), exocrine tissue and the islets of the pancreas (Fig. 2L; arrow heads). Other tissues did not show a positive reaction for ANGPTL8 (data not shown). The results of IHC are summarized in Table I.
ISH
In the liver (Fig. 3A), the cytoplasm of hepatocytes in zone 1 showed a positive signal with the anti-sense probe (Fig. 3B; arrows), whereas no signal was detected with sense probe (Fig. 3C). In the adipose tissue (Fig. 3D), the immature adipocytes showed a strong signal (Fig. 3E; arrowheads), and a weak signal was observed in mature adipocytes (Fig. 3F). In hibernoma (Fig. 3G), the tumor cells showed a positive reaction in the cytoplasm (Fig. 3H). No signal was detected in slides hybridized with the sense probe (Fig. 3I). In the section treated with RNase and hybridized with anti-sense probe (Fig. 3J) or in the section hybridized with no probe (Fig. 3K), no signals were detected. In lipoma, a weak signal was observed in mature tumor cells with the anti-sense probe, and other tissues did not show a positive reaction (data not shown). The results of ISH are summarized in Table I.
Discussion
In the present study, RT-qPCR showed that ANGPTL8 mRNA was expressed in the liver, normal adipose tissue, lipoma and hibernoma. These results are consistent with previous findings, which suggest ANGPTL8 is abundantly expressed in the liver, adipose tissue and BAT (2,5,6). The localization data of ANGPTL8-positive cells by IHC and ISH in these tissues were comparable, suggesting the specific reactivity of the antibody used in the current study. The present study revealed the precise localization of ANGPTL8-expressing cells in the liver and adipose tissue. The expression of ANGPTL8 was homogenous in hibernoma. The distribution of ANGPTL8-expressing cells in the liver and adipose tissue was heterogeneous; there was a difference in the expression levels among the cells in the organs.
In the liver, ANGPTL8 expression was observed in the periportal hepatocytes of the hepatic acinus. The hepatic acinus is separated into three functional zones, and hepatocytes in the periportal zone 1 actively uptake triglycerides and utilize them through β-oxidation (9). ANGPTL8 is expressed in hepatocytes, which actively metabolize triglycerides under physiological conditions. Increased expression of ANGPTL8 has previously been detected in cultured cells of hepatocellular carcinoma (3). The expression of ANGPTL8 may reflect the metabolic state of carcinoma cells. Serum levels of ANGPTL8 have been shown to correlate with liver steatosis (10) and non-alcoholic fatty liver disease (11). The association of the precise localization of ANGPTL8-expressing hepatocytes with lipid deposition requires to be elucidated.
In adipose tissue, ANGPTL8 was more strongly expressed in immature adipocytes compared with mature adipocytes. This is consistent with previous findings on the expression of ANGPTL8 in preadipocytes, which differentiate into mature adipocytes (2). ANGPTL8 expression may be necessary for the storage of triglycerides. Previous studies reported that the knockdown of ANGPTL8 by siRNA in cultured adipocytes does not affect differentiation, but alters the expression levels of enzymes involved in lipolysis and fatty acid oxidation, resulting in a reduction in triglyceride levels (2,12). Decreases in body weight and fatty tissue have been reported in ANGPTL8 knockout mice (7). The results in the present study further suggested an important role for ANGPTL8 in lipid metabolism by immature adipocytes.
A treatment with an ANGPTL8 antisense oligonucleotide has been reported to promote the uptake and storage of triglycerides in adipose tissue and eventually prevent liver steatosis, and this has been associated with improvements in glucose intolerance in rodents (13). In this situation, immature adipocytes may be a therapeutic target of antisense oligonucleotides against ANGPTL8. Immature adipocytes release various metabolic factors, such as leptin and adiponectin (14), and the expression of enzymes associated with the lipid metabolism and leptin are upregulated in adipocytes with ANGPTL8 knockdown (12). Therefore, it is conceivable that the prevention of steatosis and improvements in glucose metabolism was, in part, attributed to the modulation of metabolic factors by ANGPTL8 knockdown in immature adipocytes.
In the present study, we identified two splice variants of ANGPTL8 in the liver, which resulted in production of truncated forms of ANGPTL8 protein. These splice variants contain a conserved region of specific epitope 1, which is necessary for the inhibition of LPL. However, the coiled-coil domains, which are necessary for binding with ANGPTL3 (15,16), were deleted. The inhibitory effect of LPL by ANGPTL8 requires complex formation with ANGPTL3 (17-19). ANGPTL8 further forms a complex with ANGPTL4, inactivating the inhibitory effect of ANGPTL4 on LPL (19). The almost complete deletion of C-terminal coiled-coil domains of ANGPTL8, like splice variant 1, may loose the ability to form complex. The partial deletion of the C-terminal coiled-coil domain of ANGPTL8, like splice variant 2, may interfere the complex formation with ANGPTL3 and ANGPTL4 and affect the partitioning of lipids by modulating the inhibitory effect on LPL. Although the expression level of splice variant 2 was very low in the normal liver, the expression level of ANGPTL8 in metabolic and liver diseases needs to be elucidated.
The splice variants assessed in the present study contained two SNPs (rs1541922 and rs192460764). SNP rs192460764 caused the replacement of an amino acid (p.Arg172Trp). A previous study reported that another SNP rs2278426, causing the same replacement (p.Arg59Trp), is associated with ethnic and gender-specific differences in lipid levels in the Chinese population (20). The frequency of this SNP is high in patients with type 2 diabetes in Japan (21). The biological significance of SNPs of ANGPTL8 in lipid metabolism and metabolic diseases needs to be clarified.
An increase in serum levels of ANGPTL8 was reported in patients with type 1 and type 2 diabetes (22,23), as well as gestational diabetes (24). It was reported that ANGPTL8 stimulates the proliferation of β-cells in the pancreatic islet (5); however, the biological activity is not reproduced (25,26). The increase in ANGPTL8 in the serum is considered to be associated with lipid dysmetabolism in diabetes (27). A direct association between ANGPTL8 expression and β-cell proliferation was not evident in the present study.
The present study demonstrated high expression levels of ANGPTL8 and detailed the precise localization of the cells expressing ANGPTL8 in the liver and adipose tissue. The expression was associated with the activity of lipid metabolism and differentiation state in these organs. The modulation of metabolism and partitioning of triglyceride by the alteration of ANGPTL8 by anti-sense oligonucleotide may be a novel therapy for lipid dysmetabolism or steatohepatitis. The localization of ANGPTL8-expressing cells in the liver and adipose tissue may aid the understanding of partitioning of lipids and development of novel therapies targeting the lipid metabolism.
Acknowledgements
The authors would like to acknowledge the excellent assistance of Ms. Kiyoko Kawahara and Mr. Takenori Fujii for help with ISH, Mr. Kiyoshi Teduka and Ms. Yoko Kawamoto for help with IHC and Ms. Taeko Kitamura for help with the subcloning and RT-qPCR (Department of Integrated Diagnostic Pathology, Nippon Medical School, Tokyo, Japan).
Funding
No funding was received.
Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author on reasonable request.
Authors' contributions
NA, RW and ZN designed the study and wrote the manuscript. NA performed histological examinations. NA and RW conducted biochemical examinations, data analyses and statistical analyses. NA prepared the figures and table. NA and KI provided clinical data of the patients and assisted with revising the manuscript. KI and ZN supervised the experimental design and manuscript writing. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Nippon Medical School Hospital (Tokyo, Japan; approval no. 30-11-1304). Informed consent was obtained from all patients.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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