Abstract

   The small intestine is main site of exogenous lipid digestion and
   absorption, and it is important for lipid metabolic homeostasis. Cell
   death-inducing DNA fragmentation-factor like effector C (CIDEC) is
   active in lipid metabolism in tissues other than those in the
   intestine. We developed small intestine-specific CIDEC (SI-CIDEC^-/-)
   knockout C57BL/6J mice by Cre/LoxP recombination to investigate the in
   vivo effects of intestinal CIDEC on lipid metabolism. Eight-week-old
   SI-CIDEC^-/- mice fed a high-fat diet for 14 weeks had 15% lower body
   weight, 30% less body fat mass, and 79% lower liver triglycerides (TG)
   than wild-type (WT) mice. In addition, hepatic steatosis and fatty
   liver inflammation were less severe in knockout mice fed a high-fat
   diet (HFD) compared with wild-type mice fed an HFD. SI-CIDEC^-/- mice
   fed an HFD diet had lower serum TG and higher fecal TG and intestinal
   lipase activity than wild-type mice. Mechanistic studies showed that
   CIDEC accelerated phosphatidic acid synthesis by interacting with
   1-acylglycerol-3-phosphate-O-acyltransferase to promote TG
   accumulation. This study identified a new interacting protein and
   previously unreported CIDEC mechanisms that revealed its activity in
   lipid metabolism of the small intestine.

   Keywords: obesity, hepatic steatosis, small intestine, CIDEC, lipid
   absorption phosphatidic acid

Introduction

   Obesity is a global epidemic disease that seriously endangers human
   health. In 2016, the World Health Organization estimated that the
   prevalence of obesity, a body mass index ≥30 kg/m^2, was 36.2% in the
   United States, 29.4% in Canada, 29% in Australia, 28.9% in Mexico, and
   23.1% in Russia [47]^1. Obesity is caused by an imbalance of fat
   metabolism that involves the intestine. The apical membrane of the
   intestinal villi absorbs digested lipids and circulating lipids are
   transported from the blood circulation through the basal membrane for
   catabolism in the intestinal cells [48]^2^-[49]^4. Lipases break down
   exogenous lipids in the intestinal lumen into small molecules such as
   2-monoacylglycerols (MAGs), fatty acids (FAs), lipoprotein lipases
   (LPLs) and cholesterol esters (CEs). FAs can be actively transported in
   the presence of proteins such as CD36, scavenger receptor class B type
   1 (SR-B1), and fatty acid transport protein (FATP) 4, but mainly rely
   on FAs, MGs, and LPL micelles with bile acid salts to enter the
   intestinal epithelium [50]^5^-[51]^8. After entering the cell, lipid
   products of digestion are transported into the endoplasmic reticulum
   (ER) where they are re-esterified by alpha-1,3-mannosyl-glycoprotein
   2-beta-N-monoacetylglyceride acyltransferases (MGATs) and diglyceride
   acyltransferases (DGATs) to form triacylglycerols (TGs) that are used
   to form lipid droplets for storage in the cytoplasm or for chylomicron
   synthesis. Mature chylomicrons are transported by lymph into the blood
   and are a source of lipids for peripheral tissues, especially the
   liver.

   CIDEC (cell death-inducing DNA fragmentation-factor like effector C),
   also known as fat specific protein (Fsp)27, was initially found to be
   associated with the adipose differentiation of 3T3-L1 cells [52]^9. The
   CIDEC protein promotes formation of unilocular lipid droplets
   [53]^10^-[54]^12. In 2015, an Fsp27 isoform, Fsp27β/CIDEC-l, which has
   10 more amino acids than Fsp27α/CIDEC-s was described [55]^13.
   Alternative start codons are responsible for the formation of different
   splices of CIDEC [56]^13. CIDEC-l is highly expressed in steatotic
   livers. The functions of CIDEC in the liver, adipose tissue, and the
   whole body have been described [57]^11^-[58]^17, but the role of CIDEC
   in the lipid metabolism of the small intestine is not clear. We
   previously reported that the longer isoform is the one that is
   primarily expressed in the small intestine in pigs [59]^18. We
   investigated CIDEC activity in the homeostasis of lipid metabolism in
   small intestine-specific CIDEC knockout (SI-CIDEC^-/-) mice. After 14
   weeks of a high-fat diet (HFD), the knockout mice weighed less and had
   less fat than wild-type (WT) mice. Loss of CIDEC function alleviated
   diet-induced obesity and hepatic steatosis. The study revealed that
   intestinal CIDEC regulated lipid metabolism homeostasis, and provided a
   rationale for subsequent research.

Material and Methods

Animals

   Small intestine-specific knockout CIDEC mice (SI-CIDEC^-/-) were
   obtained by backcrossing CIDEC^flox/flox mice in which a loxP site is
   inserted at both ends of exon 3 of CIDEC, with Villus-Cre mice
   heterozygous for C57BL/6J as a background. SI-CIDEC^-/- mice were
   Villus-Cre mice with homozygous CIDEC^flox/flox. Homozygous
   CIDEC^flox/flox mice without Villus-Cre were used as WT control mice.
   Real-time polymerase chain reaction (RT-PCR) was performed with the
   primers 5′-AAATACTCTATGGCTGCCTCCCC-3′, 5′-TGTCTAACAAGTCCCCAAACTGT-3′ to
   separate CIDEC^flox/flox (200 bp) and the WT gene (139 bp) in mouse
   offspring with Villus-Cre. Both CIDEC^flox/flox and Villus-Cre mice
   were purchased from Cyagen (Suzhou, China). The mice were male and were
   housed at 22-24°C in a 12 h light/dark cycle. Mice from the same litter
   were randomly assigned to groups of 8-10 animals each. There were no
   significant differences in the mean body weights between the groups. To
   model obesity, mice were fed an HFD, 60% kcal from fat, produced by
   Trophic Animal Feed High-Tech Co; Haian, China). A normal diet (ND) was
   the control. Body composition was recorded once weekly for 9 weeks
   after modeling. Mice were fasted for 12 h at 22 weeks of age (i.e., 14
   weeks of modeling) and then killed by breaking their necks. Eye-vein
   blood, liver tissue, epididymal fat, and subcutaneous fat were
   collected. The animal procedures were performed following the
   guidelines, and with approval, of the animal experimental ethics
   guidelines of Guangxi University (no. GXU-2021-120).

Computed tomography (CT)

   22-week-old mice were fasted for 6 h, anesthetized by injection of
   1.25% tribromoethanol at 10 μl/g body weight, and evaluated by an X-ray
   Safety Report Skyscan 1278 (Bruker; Rheinstetten, Germany) for adipose
   tissue distribution, which was an alyzed by the company's supporting
   software to label the fat as red [60]^19.

Energy expenditure

   Energy metabolism, CO[2] production, oxygen consumption, and activity
   were measured every 5 min for 2 days with a Promethion ∞RE Metabolic
   Analysis System CGF (Sable Systems International; North Las Vegas, NV,
   USA) as previously descibed [61]^20.

Glucose tolerance (GTT) and insulin tolerance (ITT) tests

   GTTs were performed in 15-week-old mice after a 16 h fast. Mice fed an
   HFD or an ND were injected with a 20% glucose solution at 5 μL/g body
   weight, and the blood glucose concentration was measured and recorded
   at 0, 15, 30, 60, 90, and 120 min. ITTs were performed in 18-week-old
   mice after fasting for 6 h. Mice fed an HFD or ND were injected with
   insulin 0.5 U/kg body weight, and the blood glucose concentration was
   determined as for the GTT.

Histology

   Tissue was stored in 4% paraformaldehyde for 24 h, passed through a
   12%, 20%, and 30% sucrose series for 12 h per step, and embedded in
   Tissue-Tek OCT compound (Sakura; Oakland, CA, USA), and 5 µm frozen
   sections were cut at -25°C with a microtome (Leica Biosystems,
   Shanghai, China). The sections were mounted on glass slides, stained
   with hematoxylin and eosin or oil red O following the manufacturer's
   (Beyotime; Shanghai, China) protocol. The sections were cover slipped
   with resin, and photographed with a Nikon 90i microscope using a 40×
   objective.

Postprandial TG secretion

   SI-CIDEC^-/- and WT mice fed an HFD or ND were fasted overnight (16 h),
   and 10 min after injecting tyloxapol (MedChemE-xpress; Shanghai, China)
   500 mg/kg, olive oil gavage were carried out at 15 uL per gram of body
   weight, blood was collected from the tail vein of 21-week-old mice at
   0-6 h post-gavage and used to measure serum TG.

Serum and tissue biochemical analysis

Sample preparation

   Small intestinal tissue was divided into three equal segments of the
   proximal (duodenum), middle (jejunum), and distal (ileum) sections. Two
   centimeters of each section were removed, flushed with prechilled
   phosphate buffered saline and placed in RIPA buffer (Solarbio; Beijing,
   China). After lysis, the samples were centrifuged at 4 °C for 10 min at
   12 000 rpm, and the supernatant was taken for assaying. Liver tissue
   samples were prepared in the same way. Blood samples were rested at 4
   °C for 1 h and centrifuged at 3000 rpm for 20 min at 4 °C, and the
   serum supernatant was collected.

Biochemical assays

   TGs, total cholesterol (TC), serum lipase (LPS), alanine transaminase
   (ALT), aspartate aminotransferase (AST), high-density lipoprotein
   cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C)
   were assayed with commercially available kits (Njjcbio; Nanjing, China)
   following the manufacturer's instructions. Malonaldehyde (MDA) was
   assayed with a lipid peroxidation kit and protein concentration was
   assayed with a bicinchoninic acid assay kit (both from Beyotime;
   Shanghai, China). Intestinal tissue phosphatidic acid was assayed with
   a mouse ELISA kit (Coiba Bio; Shanghai, China). Optical density was
   measured with an Infinite M200 Pro microplate reader (Teacan;
   Männedorf, Switzerland). All values were normalized to protein
   concentrations.

IPEC-J2 intestinal porcine epithelial cells

   IPEC-J2 cell were cultured in complete Dulbecco's minimal Eagle medium
   (DMEM containing 1% cyanine-streptomycin and 10% fetal bovine serum and
   high-fat induction by DMEM containing 0.4% oleic acid, 0.2% palmitic
   acid, 1% cyanine-streptomycin, 2% bovine serum albumin at 37 °C and 5%
   CO[2]. Cells were transfected with pcDNA3.1(-)-CIDEC-l (sus) plasmids
   or empty vectors were mixed with liposome following the manufacturer's
   protocol (Yesen; Shanghai, China). The liposomes complexes were
   transfected into IPEC-J2 cells cultured in serum-free DMEM. After 4-6
   h, the medium was changed to OAPA. Cell samples were collected after 48
   h. Cell lysates were prepared using RIPA buffer as described for
   tissues.

RNA isolation and RT-PCR

   RNA was isolated with Trizol reagent (Invitrogen; Solarbio; Beijing,
   China). cDNA was reverse transcribed from 1 µg RNA by M-MLV reverse
   transcriptase (Promega, Beijing, China). RT-PCR assays were performed
   using Real Star Green Fast mixture with ROXII (GeneStar; Beijing,
   China) and a qTOWER3G thermal cycler (Analytik; Jena, Germany). The
   primer sequences are listed in [62]Table S1. For statistical analysis
   of mRNA expression, values were calculated using the 2^-ΔΔCT method.

16S rRNA sequencing of gut microbes

   Fresh feces obtained from six 22-week-old mice fed an HFD were used.
   The full length 16S gene (27bp-1492bp) was amplified using the primers:
   5′-AGAGTTTGATCCTGGCTCAG-3′; 5′-AGAGTTTGATCCTGGCTCAG-3′. Sequencing and
   statistical analysis were performed by Biozeron (Shanghai, China).

Western blotting

   Protein supernatant concentration was measured by the bicinchoninic
   acid method. Samples were added to protein loading buffer and boiled
   for 10 min at 100 °C. Anti-CIDEC (1:2000; Cloud-Clone Corp; Wuhan,
   China) and anti-alpha tubulin (Beyotime; Shanghai, China) were the
   primary antibodies. The blots were developed using HRP-conjugated
   secondary antibodies (1:3000) and an enhanced chemoluminescence-plus
   system (Bio-rad; California, USA).

Co-immunoprecipitation and mass spectrum (Co-IP/MS)

   Small intestine tissue was collected from WT mice (no flox). Intestinal
   protein supernatants were prepared and the protein concentration was
   assayed as described above. The supernatants were immunoprecipitated
   with a polyclonal CIDEC antibody (Cloud-Clone Corp; Wuhan, China)
   following the manufacturer's protocol. Briefly, 3 mg protein
   supernatant samples were precleared by IgG and arotein L-agarose (Santa
   Cruz Biotechnology; Dallas, TX, USA) for 30 min, incubated with 2 mg of
   primary antibody for 1h and then incubated with protein L-agarose at 4
   °C overnight (16 h). The bound proteins were washed twice with
   prechilled RIPA and eluted by boiling in Sodium dodecyl-sulfate
   polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Solarbio;
   Beijing, China). The resulting proteins were assayed by SDS-PAGE and
   sent to APTBIO (Shanghai, China) for mass spectrum analysis.

Kyoto Encyclopedia of Genes and Genomes (KEGG) and genome database enrichment
analysis

   The proteins identified by mass spectrum were included in a FASTA
   database that was used for KEGG pathway enrichment analysis, which was
   performed by the online biological tool KOBAS 3.0.

Statistical analysis

   The results were reported as means ± standard error of the mean (SEM)
   analyzed by Excel using unpaired t-tests or One-way analysis to
   calculate significance. P-values < 0.05, < 0.01, and < 0.001 were
   considered significant.

Results

SI-CIDEC^-/- mice were resistant to diet-induced obesity

   To obtain SI-CIDEC^-/- mice, loxP sites were inserted at both ends of
   exon 3 of CIDEC in CIDEC^flox/flox mice (Figure [63]1A), primers on
   both ends of the first loxP site were used for PCR, and agarose gel
   electrophoresis was used to distinguish heterozygous from homozygous
   CIDEC^flox/flox mice (Figure [64]1B). PCR identified CIDEC with a loxP
   site of 200 bp, while that of the WT CIDEC was 139 bp. CIDEC^flox/flox
   mice were crossed with Villus-Cre mice to generate small
   intestine-specific (SI-CIDEC^-/-) mice. In SI-CIDEC^-/- mice, exon 3 of
   CIDEC, consisting of 154 bp, was knocked out, which caused a code-shift
   mutation and both two isoforms of CIDEC were knockout. As a result,
   CIDEC protein was not detected in SI-CIDEC^-/- mouse intestine, but no
   change was seen in the liver (Figure [65]1C). To model obesity,
   SI-CIDEC^-/- and WT mice were fed an HFD and controls were fed an ND
   (Figure [66]1D). As shown in (Figure [67]1E-G), SI-CIDEC^-/- mice had
   significantly lower mean body weight and fat mass than WT mice on the
   HFD at 6 weeks post-modeling. At 14 weeks after HFD induction, the
   SI-CIDEC^-/- mice had reduced body size and fat mass (Figure [68]2A,
   B). Body composition analysis showed that SI-CIDEC^-/- -HFD mice had
   15% lower body weight and 30% lower fat mass compared to WT-HFD mice
   (Figure [69]2C-E). Consistent with those results, the weights of
   subcutaneous and epididymis adipose tissue were reduced by 23.4% and
   25.1% respectively (Figure [70]2F, G). No difference was found in
   adipocyte size in SI-CIDEC^-/--HFD and WT-HFD mice (Figure [71]2H, I).
   Interestingly, SI-CIDEC^-/--ND and WT mice had similar fat masses, but
   adipocyte size was reduced by 37.07% in the SI-CIDEC^-/--ND mice
   compared with the latter. Subsequently, we measured the mRNA expression
   of inflammation-related genes in adipose tissue. The results showed
   that the mRNA expression of both IL-6 and Nos2 was significantly
   decreased (Figure [72]2J). As in CIDEC-deficient mice [73]^11, glucose
   tolerance testing found that SI-CIDEC^-/--HFD mice had increased
   glucose tolerance compared with those fed an ND (Figure [74]2K).
   Differences in the insulin tolerance test results obtained in the
   SI-CIDEC^-/- and WT mice fed either the HFD or ND were not significant
   (Figure [75]2L).

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   Generation and modeling of small intestine-specific knockout CIDEC
   mice. (A) Genotyping strategy of CIDEC^flox/flox and SI-CIDEC^-/- mice.
   (B) Genotype identification by AGE (agarose gel electrophoresis) for
   distinguishing mice with homozygous CIDEC^flox/flox from wild-type
   CIDEC (no flox) and heterozygous CIDEC^flox/flox; sites of primers are
   shown in Fig [78]1A; (C) Western blots of CIDEC protein of the liver
   and small intestine in SI-CIDEC^-/- WT mice; (D) Workflow of
   establishing high-fat model mice. (E-G) Body composition of mice during
   0-9 weeks after modeling, mice modeling from 8 weeks of age; WT mice
   fed a normal diet (WT-ND, n=10), SI-CIDEC^-/- mice fed an ND
   (SI-CIDEC^-/--ND, n=7), WT mice fed a high-fat diet (WT-HFD, n=7) and
   SI-CIDEC^-/- mice fed a high-fat diet (SI-CIDEC^-/--HFD, n=7) were
   used. Data are means ± SEM. Differences of WT versus SI-CIDEC^-/- mice
   fed an HFD were considered significant at *p< 0.05, **p< 0.01.

Figure 2.

   [79]Figure 2
   [80]Open in a new tab

   SI-CIDEC^-/- mice were resistant to diet-induced obesity. (A-K)
   22-week-old mice fed high-fat or normal diets mice were modeled
   beginning at 8 weeks of age used. (A) Representative photographs of
   mice; Scale bar: 1cm; (B) Representative computed tomography (CT) scan
   analysis. Fat is shown in red; (C-E) Body composition (n=7-10); (F)
   Representative photographs of adipose tissue; Scale bar: 1 cm; (G)
   Weight of subcutaneous and epididymal adipose tissue (n=7-10); (H)
   Representative photographs of adipose tissue histology and (I)
   adipocyte area; stained by hematoxylin and eosin. Scale bar: 100 µm
   (40× objective); (J) mRNA of inflammation-related genes, n=3-4; (K)
   Serum glucose concentrations of 15-week-old mice in the glucose
   tolerance tests (GTTs) (n=6-10); Differences were considered
   significant at *p < 0.05. (L) Serum glucose concentrations of
   18-week-old mice in insulin tolerance tests (ITTs, n=6-10). Data are
   means ± SEM. Mean values with different superscript letters differ
   significantly (p < 0.05).

SI-CIDEC^-/- improved diet-induced hepatic steatosis and hyperlipidemia risk

   As obesity often causes fatty liver, we investigated the effects of
   gut-specific knockout of CIDEC on HFD-induced fatty liver. Mice fed
   high-fat diets were larger and heavier than those fed an ND and liver
   tissue pathology was observed, WT-HFD mice had severe hepatic steatosis
   (Figure [81]3A-C), and the steatotic changes in SI-CIDEC^-/--HFD mice
   were not different from those in SI-CIDEC^-/--ND and WT-ND mice.
   Compared with WT-HFD mice, SI-CIDEC^-/--HFD mice had significantly
   reduced liver TG, TC, and MDA levels that were similar to the those in
   mice fed an ND (Figure [82]3D, E). In addition, SI-CIDEC^-/--HFD mice
   had significantly lower serum ALT than WT-HFD mice. Serum AST was also
   lower in SI-CIDEC^-/--HFD mice than in WT-HFD mice, but the difference
   was not significant (Figure [83]3G, H). The HFD increased serum TG, TC
   and LDL-C in WT mice (Figure [84]3H, I). Serum LDL-C was positively
   correlated with the risk of cardiovascular disease. As the
   SI-CIDEC^-/--HFD mice had lower TG, TC and LDL-C levels than the WT-HFD
   mice, small intestine-specific knockout of CIDEC may reduce
   cardiovascular disease risk by improving blood cholesterol composition.
   The mRNA expression of inflammation-related genes (IL-6, TNF-α, Ccl2,
   and Ccl5) was decreased, and the mRNA expression of lipid
   homeostasis-related genes (LXRα and DGAT2) was increased in the liver
   (Figure [85]3J), indicating that the secretion of very low-density
   lipoprotein was increased, and that the reason for the decrease of
   serum TG was decreased chylomicron (CM) secretion by the small
   intestine. The results indicate that SI-CIDEC^-/- mice resisted
   diet-induced hepatic steatosis and changes in blood cholesterol.

Figure 3.

   [86]Figure 3
   [87]Open in a new tab

   SI-CIDEC^-/- improved diet-induced hepatic steatosis and hyperlipidemia
   risk. (A-I) 22-week-old HFD/ND-fed mice (n=5-10) modeling from 8-week
   age was used. (A) Representative photographs of liver tissue; Scale
   bar: 1cm; (B) Liver weight; (C) Representative photographs of liver
   histology; Scale bar:100 µm (40× objective); (D, E) Liver TG
   (triglyceride), TC (total cholesterol) and MDA (malondialdehyde)
   concentrations; (F-I) Serum ALT (Alanine aminotransferase), AST
   (Aspartate aminotransferase), TG, TC and LDL-C (Low-density lipoprotein
   cholesterol) concentrations (n=4-10). Data are mean ± SEM. Mean values
   with different superscript letters differ significantly (p < 0.05). (J)
   mRNA expression of enzymes involved in liver lipid homeostasis and
   inflammation-related genes; SI-CIDEC^-/- and WT (n=3-4) on HFD were
   used; Data are normalized to GAPDH and mean ± SEM. Differences were
   considered significant at *p < 0.05.

Energy metabolism in SI-CIDEC^-/- mice

   The energy expenditure in mice fed an HFD and housed in metabolic cages
   was measured as previously described [88]^21. The night time O[2]
   consumption, CO[2] production, and energy metabolism of SI-CIDEC^-/-
   mice were significantly decreased compared with WT mice (Figure [89]4A,
   B, D), but differences in the respiratory exchange and predicted
   metabolic rates in the two genotypes were not significant (Figure
   [90]4C, E). However, CO[2] production, O[2] consumption, and energy
   consumption was decreased in SI-CIDEC^-/--ND mice compared with the
   WT-ND mice ([91]Figure S1A-E). The results indicate that energy
   metabolism was not the reason for improved obesity and fatty liver in
   the small intestine-specific knockout mice.

Figure 4.

   [92]Figure 4
   [93]Open in a new tab

   Energy metabolism in SI-CIDEC^-/- mice. (A-D) 17-week-old mice fed a
   high-fat diet from 8 weeks of age were used (both n = 4), data obtained
   during 2 days are shown. Each bar on the right was the mean during the
   light, night and whole day (light and night). (A) Whole body oxygen
   consumption (mL/h). (B) CO[2] production (mL/h); (C) Respiratory
   exchange ratio (VCO[2]/VO[2]); (D) Energy expenditure (Kcal/h). Data
   are means ± SEM; (E) Predicted metabolic rate (Kcal/h/40 g), the method
   has been previously predescribed. (21) Data are means ± SEM;
   Differences were considered significant at *p < 0.05, **p < 0.01, and
   ***p < 0.001.

SI-CIDEC^-/- restricted intestinal absorption of lipids

   To investigate whether the improvement of diet-induced obesity and
   fatty liver in SI-CIDEC^-/- mice was caused by altered intestinal
   digestion and absorption, we monitored food intake, fecal TG, and TC.
   They were significantly increased in SI-CIDEC^-/--HFD mice compared
   with WT-HFD mice (Figure [94]5A, B), and were not changed in ND mice.
   We modeled the postprandial state with an olive oil gavage after
   injection of tyloxapol, which showed that serum TG was significantly
   lower in SI-CIDEC^-/- mice than in WT mice, regardless of the diet
   (Figure [95]5C), consistent with reduced intestinal absorption in
   SI-CIDEC^-/-. To determine how knockout of CIDEC impaired lipid
   absorption, we investigated changes in lipid accumulation, catabolism,
   and secretion. TG and TC in tissue sections from the middle and distal
   intestine were lower in SI-CIDEC^-/--HFD that in WT-HFD mice (Figure
   [96]5D, E and [97]Figure S2A). Assays of lipase activity found
   increased lipolysis in the proximal and middle intestine of
   SI-CIDEC^-/- mice (Figure [98]5F), which may have been partly
   responsible for the reduced accumulation of intestinal lipids. In
   addition, lipase activity decreased from proximal to distal sections of
   the intestine of SI-CIDEC^-/--HFD mice and intestinal TG increased
   gradually from proximal to distal sections. Interestingly, changes in
   free fatty acid (FFA) levels in the intestine did not increase with
   increased lipase activity. Reduced FFAs were seen in the distal
   intestine (Figure [99]5G), and FFA levels in the circulating and liver
   were significantly elevated (Figure [100]5H, I).

Figure 5.

   [101]Figure 5
   [102]Open in a new tab

   SI-CIDEC^-/- restricts intestinal absorption of lipids. (A-I)
   22-week-old mice fed high-fat (HFD) or normal diets (n=5-10) beginning
   at 8 weeks of age. (A) Food intake of WT-ND (n=3), SI-CIDEC^-/--ND
   (n=3), WT-HFD (n=4) and SI-CIDEC^-/--HFD (n=3); (B) Fecal TG
   (triglyceride) and TC (total cholesterol) concentrations (n=4-5); (C)
   Postprandial serum TG concentrations in 0-6 h post-gavage with
   inhibition of lipase by tyloxapol (each group n=7); (D-E) Proximal,
   middle, and distal intestine in 22-week-old HFD fed mice (n=6-10) were
   used; (D) TG concentrations; Scale bar:100 μm (40× objective); (E) TC
   concentrations; (F) Lipase activity; (G) Intestinal free fatty acid
   (FFA) concentration; (H) Serum FFA concentration; (I) Hepatic FFA
   concentration; Data are means ± SEM. Differences were considered
   significant at *p < 0.05, **p < 0.01, ***p < 0.001.

   We also examined changes in the mRNA expression of genes involved in
   lipid absorption (NPC1L1, SR-B1, and CD36), transport (FATP1 and
   FATP4), synthesis (DGAT2), secretion (APOB), catabolism (ATGL, HSL, and
   MGL) and metabolism (AMPKα and PPARα) in the intestine. Compared with
   WT-HFD mice, SI-CIDEC^-/--HFD mice had a significant increase in NPC1L1
   transcripts and a decrease in monoglyceride lipase (MGL) transcripts in
   the proximal intestine ([103]Figure S2B). increased SR-B1 and FATP1 and
   decreased MGL transcripts in the middle intestine ([104]Figure S2C),
   and decreased SR-B1 and APOB in distal intestinal transcripts
   ([105]Figure S2D). The data suggest that intestinal transport of
   cholesterol and FAs increased.

   We tested the tight junction protein—ZO-1 protein expression in the
   intestine of two gene type mice under HFD. The results ([106]Figure
   S3B) showed that ZO-1 protein expression was unchanged between two
   group. The permeability test also showed that overexpression of CIDEC-l
   did not affect the permeability of IPEC-J2 cells ([107]Figure S3C). We
   conclude that there was no significant effect on intestinal
   permeability when changing CIDEC.

   To detect the effect of intestinal CIDEC on gut microbiome, we examined
   changes of the microbiome in fresh feces from mice with 16S rRNA gene
   sequencing. No significant differences in alpha diversity were found
   following analysis with the ACE, Chao1, and Shannon indexes
   ([108]Figure S4A-C). The two groups showed a separation along the PC2
   axis of a principal coordinate analysis (PCoA) plot ([109]Figure S4D).
   but no significant difference was obeserved in beta diversity of the
   two groups. Changes in the mean percentages of the intestinal
   microbiota at the phylum level or in the the Firmicutes to
   Bacteroidetes ratio were not significant ([110]Figure S4E, F). Lefse
   analysis found significant differences in 15 species of bacteria in the
   two groups ([111]Figure S4G, H), mainly belonging to the Firmicutes,
   Bacteroidetes and Proteobacteria. The results suggest that improvements
   in diet-induced obesity and hepatic steatosis did not result from
   changes of the gut microbiome.

SI-CIDEC^-/- repressed lipid deposition by inhibiting AGPAT activity

   RT-PCR showed that long isoform, CIDEC-1, was the one that was
   primarily expressed in the intestine of WT-HFD mice (Figure [112]6A),
   which is similar to the findings in experimental pig models [113]^18.
   In IPEC-J2 cells overexpressing CIDEC-l, the longer isoform of porcine
   CIDEC, treatment with oleic acid and palmitic acid (i.e. high-fat
   induction) resulted in IPEC-J2 with increased TG and TC and in lower
   extracellular TG than seen in cells transfected with empty vector
   (Figure [114]6B-C). Lipase activity was reduced (Figure [115]6D), and
   expression of ATGL mRNA was detected in IPEC-J2 cells transfected with
   CIDEC-l (Figure [116]6E).

Figure 6.

   [117]Figure 6
   [118]Open in a new tab

   CIDEC promotes lipid deposition by enhancing AGPAT enzyme activity. (A)
   mRNA expression of two isoforms of CIDEC (n=3-4); 22-week-old mice
   modeled by feeding a high-fgat diet from 8 weeks of age were used.
   (B-E) After the introduction of CIDEC-l or vector, IPEC-J2 cells
   induced by high-fat for 48 hours were used (n=3-4); (B) Intracellular
   and extracellular triglyceride (TG) concentration; (C) Intracellular
   and extracellular total cholesterol (TC) concentration; (D) Lipase
   activity; (E) mRNA expression of enzymes involved in lipid absorption
   (NPC1L1, SR-B1, CD36), transportation (FATP1, FATP4), synthesis
   (DGAT2), secretion (MTP, APOB), lipolysis (ATGL, HSL, MGL) and
   oxidative metabolism (AMPKα, PPARα) in cells normalized to GAPDH; (F)
   SDS-PAGE stained by Coomassie brilliant blue. Protein
   immunoprecipitated from total intestinal protein of wild-type mice (no
   flox) fed a normal diet. Input, total intestinal protein; control IgG,
   immunoprecipitated protein using negative control mouse IgG antibody;
   CIDEC, immunoprecipitated protein using CIDEC antibody. (G) Intestinal
   CIDEC-interacting protein enrichment KEGG analysis; (H) Phosphatidic
   acid concentrations in the middle and proximal intestine of mice fed a
   high-fat diet (n=3-5). Data are means ± SEM. Differences were
   considered significant at *p < 0.05, ***p < 0.001. (I) Mechanism of
   CIDEC accelerating TG synthesis by the glycerol-3-phosphate pathway in
   the small intestine.

   To further investigate how CIDEC affected lipid absorption in the
   intestine, we isolated proteins that interact with CIDEC (Figure
   [119]6F), and identified them by coimmunoprecipitation and mass
   spectrometry (Co-IP/MS). KEGG database enrichment analysis identified
   one of the proteins (AGPAT) in the lipid digestion and absorption
   pathway (Figure [120]6G). Because the lipid levels changed in the mid
   and distal intestine of mice fed an HFD, we assayed phosphatidic acid
   concentrations in those two sections. The data indicated that the
   concentration of phosphatidic acid was significantly lower in the
   SI-CIDEC^-/--HFD mice than in WT-HFD mice (Figure [121]6H). AGPAT is
   abundant in the ER and catalyzes the conversion of lysophosphatidic
   acid to phosphatidic acid in the glycerol-3-phosphate pathway. After
   being dephosphorylated by PAP, phosphatidic acid is transformed into
   diacylglycerol (Figure [122]6I). As the glycerol-3-phosphate pathway
   accounts for 20-30% of TG synthesis during lipid absorption
   [123]^22^-[124]^25, which suggests SI-CIDEC^-/- decreased TG
   accumulation by inhibiting the catalytic activity of AGPAT.

Discussion

   Previous studies reported aggravated liver steatosis associated with
   increased serum TG in CIDEC-deficient, CIDEC^-/-/ob/ob
   double-deficient, CIDEC^-/-/BATless, and adipose-specific knockout mice
   [125]^12^-[126]^17. The evidence suggests that CIDEC is required for
   adipose tissue absorption and storage of circulating lipids. CIDEC
   expression in visceral adipose tissue was significantly decreased in
   obese or obese-T2D subjects compared with nonobese subjects [127]^26^,
   [128]^27. Here, we found that CIDEC protein expression in the intestine
   was reduced by the HFD ([129]Figure S3A), analogous changes in CIDEC
   expression in the adipose tissue and the small intestine suggesting
   that CIDEC may play similar roles in those tissues. Our results showed
   that SI-CIDEC^-/- mice fed an HFD weighed less, had a smaller fat mass,
   much like previous studies, but the difference was that SI-CIDEC^-/-
   mice had normal livers without steatosis and without change in energy
   metabolism compared with WT mice fed an HFD. Intestinal CIDEC may thus
   be a suitable target for the treatment of obesity, with reduced lipid
   accumulation in critical metabolic tissues.

   Impaired intestinal lipid absorption was shown by increased fecal TG
   concentration and decreased postprandial serum TG concentration in
   SI-CIDEC^-/- mice. In addition, no changes in liver, serum and fecal TG
   and TC concentrations of SI-CIDEC^-/--ND compared with WT-ND mice were
   seen. Reduced food intake and energy metabolism in SI-CIDEC^-/--ND mice
   were responsible for its decreased adipocyte size but unchanged adipose
   weight, fat mass, or body weight. Co-IP/MS revealed that AGPAT was a
   potential downstream target of CIDEC, CIDEC accelerated the synthesis
   of TG from G3P by enhancing the AGPAT activity.

   As no change in the energy metabolism of SI-CIDEC^-/- mice fed an HFD
   was observed, impaired lipid absorption is most likely the main reason
   for improved obesity. What caused impaired lipid absorption in the
   small intestine of SI-CIDEC^-/- mice? Cholesterol, a precursor of bile
   acids, was reduced in SI-CIDEC^-/--HFD mice. Reduced bile acid
   secretion may be one of the reasons for the decreased lipid absorption
   that was observed. The digestion and absorption of lipids cannot be
   achieved without lipase, and inhibition of CIDEC has been reported to
   enhance ATGL activity, thus increasing lipolysis [130]^3^, [131]^28^,
   [132]^29. The absence of intestinal ATGL was shown not affect lipid
   absorption and secretion in mice [133]^3, but small intestine-specific
   overexpression of ATGL in mice has been shown to decrease intestinal TG
   without affecting the secretion of TG chylomicrons or changing body
   weight [134]^30. The evidence shows that ATGL is not necessary for
   intestinal absorption in mice, and suggests that increased lipase
   activity in the intestine of SI-CIDEC^-/- mice was not directly
   responsible for the inhibition of intestinal absorption. As
   MGL-deficient mice have reduced feed conversion [135]^31^, [136]^32,
   decreased MGL expression in the SI-CIDEC^-/- mouse intestine could
   affect lipid absorption. Lipid absorption is complicated and involves
   multiple pathways that could be influenced by intestinal CIDEC
   knockout.

   Co-IP/MS identified AGPAT as a target protein of CIDEC in the
   regulation of intestinal lipid metabolism and homeostasis. Previous
   studies have shown that AGPAT1-deficient mice die before weaning and
   have reduced body weight, fat mass, and blood glucose [137]^33.
   AGPAT2-deficient mice have congenital generalized lipodystrophy,
   develop severe fatty liver even if fed an ND, and do not form white and
   brown adipose tissue because of impaired adipose differentiation
   [138]^34^, [139]^35. Downregulation of PPARγ and C/EBPβ transcription
   has been detected in AGPAT2-deficient mice, and CIDEC expression is
   known to be regulated by transcription factors including SREBP1c,
   PPARγ, C/EBP, and PPARα [140]^36^-[141]^39. Both AGPAT1 and AGPAT2
   catalyze the synthesis of TG in the glycerol-3-phosphate pathway, and
   are expressed in the small intestine. In SI-CIDEC^-/- mice fed an HFD,
   the synthesis of phosphatidic acid, which is catalyzed by AGPAT, was
   significantly reduced, suggesting the interaction of CIDEC and AGPAT.
   The study found that CIDEC promoted lipid absorption in the mouse
   intestine that affected the homeostasis of systemic lipid metabolism.
   Small intestine-specific knockout of CIDEC improved but did not
   completely impair lipid absorption in mice by inhibiting AGPAT
   activity. Intestinal CIDEC may be a novel target for the treatment of
   obesity-associated diseases.

Supplementary Material

   Supplementary figures and table.
   [142]Click here for additional data file.^ (957.1KB, pdf)

Acknowledgments