Abstract
The incorporation of plasma triglyceride (TG) fatty acids to white adipose tissue (WAT) depends on lipoprotein lipase (LPL), which is regulated by angiopoietin-like protein-4 (ANGPTL-4), an unfolding molecular chaperone that converts active LPL dimers into inactive monomers. The production of ANGPTL-4 is promoted by fasting and repressed by feeding. We hypothesized that the postprandial hormone cholecystokinin (CCK) facilitates the storage of dietary TG fatty acids in WAT by regulating the activity of the LPL/ANGPTL-4 axis and that it does so by acting directly on CCK receptors in adipocytes. We report that administration of CCK-8 (a bioactive fragment of CCK) to rats: (i) reduces plasma ANGTPL-4 levels; (ii) represses Angptl-4 expression in WAT and (iii) simultaneously enhances LPL activity in this tissue without inducing Lpl expression. In vivo CCK-8 effects are specifically antagonized by the CCK-2 receptor (CCK-2R) antagonist, L-365,260. Moreover, CCK-8 downregulates Angptl-4 expression in wild-type pre-adipocytes, an effect that is not observed in engineered pre-adipocytes lacking CCK-2R. These effects have functional consequences as CCK-8 was found to promote the uptake of dietary fatty acids by WAT, as demonstrated by means of proton nuclear magnetic resonance (1H-NMR). The efficacy of acute CCK-8 administration was not reduced after chronic CCK-8 treatment. Moreover, the effects of CCK-8 on WAT were not associated to the increase of circulating insulin. Our results show that cholecystokinin promotes lipid storage in WAT by acting on adipocyte CCK-2R, suggesting a pivotal role for CCK in TG homeostasis.
Introduction
Cholecystokinin (CCK) is a postprandial hormone secreted by mucosal epithelial cells in response to fat in the lumen of the duodenum. Direct effects of CCK include satiety, inhibition of gastric emptying and stimulation of lipase release by the exocrine pancreas (Owyang & Logsdon 2004). Moreover, CCK indirectly activates fat catabolism by cooperating both centrally and peripherally with leptin (Morton et al. 2005, Merino et al. 2008a , Montgomery et al. 2013, Irwin et al. 2013a ) and by modulating leptin release from adipocytes (Attoub et al. 1999). CCK binds two different receptors belonging to the G protein-coupled receptor family called CCK-1 receptor (CCK-1R), located in different organs of the digestive system and in some areas of the central nervous system (CNS), such as the brainstem and the hypothalamus and CCK-2R, widely distributed throughout the CNS (Cano et al. 2003, Dufresne et al. 2006). The relevance of CCK in energy metabolism is also supported by data from OLETF rats, which lack functional CCK-1 receptors (CCK-1R) and exhibit marked obesity and type II diabetes (Moran & Bi 2006, Schroeder et al. 2009), and CCK-knockout mice that, in contrast, are resistant to high-fat diet (HFD)-induced obesity (Lo et al. 2010). In addition, recent studies have shown that therapies based on CCK analogues alone or in combination with leptin reduce plasma triglyceride (TG) and LDL levels and also limit ectopic lipid deposition in insulin-resistant animal models (Irwin et al. 2013a , b ). Finally, we previously demonstrated that acute administration of the sulphated C-terminal octapeptide of CCK (CCK-8) to free-feeding rats reduces food intake and enhances enzymatic mechanisms compatible with increased catabolic activity in skeletal muscle (Merino et al. 2008a ).
Together, these data support the concept that CCK is involved in regulating TG homeostasis. This regulation would be accomplished by: (i) limiting food intake; (ii) delaying gastric emptying; (iii) facilitating intestinal digestion of fat and TG absorption and (iv) enhancing fatty acid (FA) catabolism in oxidative tissues. More recently, King and coworkers demonstrated that FA uptake by adipose tissue in CCK-knockout mice is impaired (King et al. 2015, Weng et al. 2017). In a recent study, Demenis et al. (2017) reported that CCK-8 promotes CD-36-mediated FA uptake by mouse enterocytes. Based on these precedents, we hypothesized that CCK regulates TG uptake by WAT through activation of CCK receptors located in WAT. The confirmation of this hypothesis would allow one to speculate about a complementary role between CCK and insulin in regulating postprandial TG homeostasis (Czech et al. 2013). Therefore, the objective of our study was to identify the adipocyte as a target for CCK and to determine whether this is relevant in regulating TG homeostasis. As lipoprotein lipase (LPL) drives FA uptake in WAT, we wanted to further investigate the role of adipocyte CCK receptors in regulating the activity of the LPL-angipoietin-like protein 4 (ANGPTL-4) axis, which is pivotal in the uptake of plasma TG by WAT (Sukonina et al. 2006). ANGPTL-4 is a molecular chaperone that binds and inactivates LPL by promoting the dissociation of the LPL homodimer, which is the enzymatically active form of LPL. ANGPTL-4 is emerging as a new class of adipokine that appears to be regulated by nutritional status and hormones, such as insulin and glucocorticoids (Kadomatsu et al. 2011).
In this study, the effect of CCK-8, one of the most abundant circulating bioactive forms of CCK that preserves the biological activity of the native full-length hormone (Cantor & Rehfeld 1989), was characterized both in vivo and in vitro. First, the effects of CCK-8 on ANGPTL-4 and LPL expression, LPL activity and fatty acid uptake, as determined by proton nuclear-magnetic resonance (1H-NMR), were characterized in rat WAT. Second, CCK-8’s effects were corroborated in pre-adipocytes obtained from rat visceral and subcutaneous WAT. The specificity of effects of CCK was assessed by means of (i) in vivo-administered CCK receptor antagonists and (ii) pre-adipocytes in which CCK receptors were silenced.
Material and methods
Chemicals
The CCK-1R antagonist, SR-27,897 (1-[2-4-(2-chlorophenyl)thiazol-2-yl-aminocarbonyl]-indolylacetic acid), was kindly provided by Sanofi-Synthelabo, France (Poncelet et al. 1993). The CCK-2R antagonist, L-365,260 [(3R-(+)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzo diazepin-3-yl)-N′-(3-methylphenyl)urea], was a gift of Merck Sharp and Dohme Research Laboratories, USA (Chang et al. 1989). Other chemicals were from Sigma.
Experimental design
The studies presented here conform to the Guide for the Care and Use of Laboratory Animals (European Communities Council Directive 86/609/EEC) and were approved by the Ethics Committee of the Universidad San Pablo-CEU. The in vivo studies were carried out in adult male Sprague–Dawley rats during the nocturnal period of the circadian cycle and under similar experimental conditions to those used in our laboratory to investigate the physiological role of CCK on other aspects of energy metabolism (Merino et al. 2008a , b ).
Experiment 1: Acute treatment with CCK-8
Thirty-six 14-week-old male Sprague-Dawley rats (Harlan, Barcelona, Spain) were housed individually under standard conditions (12 h light/12 h dark, 22°C; lights on at 08:00) on corncob bedding with standard rodent chow (Teklad; Harlan) and water ad libitum (day 14). In order to minimize the number of rats, the experiment was organized in three sequential assays that were carried out at 1-week intervals in the same animals, which were randomly assigned in each assay to either intervention or control groups. As schematized in Fig. 1, the assays performed were: (1) a dose–response effect of CCK-8, in order to identify the dose of CCK-8 able to modulate plasma ANGPTL-4 levels (day 0); (2) an antagonism experiment, aimed at identifying the CCK receptor subtype involved in the effect of CCK-8 (day 7) and (3) an antagonism experiment carried out in rats that acutely consumed a diet containing elevated amounts of linolenic and linoleic acids (LIN), which were used as a probe to trace dietary TGs.
Schematic illustration of acute and chronic treatment with CCK-8. (1) Acute treatment with CCK-8: The experiment was organized in three sequential assays: day 0) dose–response effect of CCK-8; day 7) antagonism of the effect of CCK-8 by selective CCK-1 (SR-27,897) and CCK-2 receptor antagonists (L-365,260) and day 14) antagonism of the effect of CCK-8 by L-365,260 in rats that consumed a diet containing elevated amounts of LIN. On days 0, 7 and 14, rats were fasted from 09:00 until 18:00 and then fed ad libitum either with standard chow (days 0 and 7) or with LIN-enriched food (day 14). On day 0, rats received CCK-8 at 22:00 and a sample of blood was collected at 24:00. Both food intake and BW variation between 22:00 and 24:00 were monitored. On day 7, animals received either L-365,260 or SR-27,897 at 21:30 and a dose of CCK-8 at 22:00. Similarly, on day 14, rats received L-365,260 and CCK-8 and were killed at 24:00. White adipose tissues were collected to quantify LIN by 1H-NMR. (2) Chronic treatment with CCK-8: CCK-8 was administered twice a day (09:00 and 18:00) during 11 days to free feeding rats. On day 12, rats received the same pharmacological treatment but were fasted from 09:00 until 18:00, then fed ad libitum either with standard chow or with LIN-enriched food, and killed at 24:00.
Citation: Journal of Endocrinology 236, 3; 10.1530/JOE-17-0580
In assay 1, rats were randomly assigned to groups receiving treatment with either saline or CCK-8 (saline, 0.025, 0.5 and 10 μg/kg; n = 6/group). The day of the experiment, rats were fasted from 09:00 until 18:00 and then fed ad libitum with standard chow. At 22:00, rats were weighed and administered (i.p.) either saline or CCK-8. After 2 h, food intake and BW were determined and blood samples were obtained by incision of the tail vein. The wound was then washed with antimicrobial solution of povidone-iodide. A pair-feeding assay, where rats were restricted to the amount of food consumed by rats receiving 10 μg/kg CCK-8, was carried out to explore the eventual contribution of reduced food intake to the effect of CCK-8 (control and pair-fed groups, n = 6/group).
In assay 2, animals were randomly re-assigned to a similar feeding protocol. In this case, at 21:30, animals received saline (n = 12), L-365,260 (a CCK-2R antagonist; 1 mg/kg; n = 12) or SR-27,897 (a CCK-1R antagonist; 0.3 mg/kg; n = 12) s.c. Thirty minutes later, rats were treated i.p. with either saline (saline + saline, n = 6; L-365,260 + saline, n = 6; SR-27,897 + saline, n = 6) or 10 μg/kg CCK-8 (saline + CCK-8, n = 6; L-365,260 + CCK-8, n = 6; SR-27,897 + CCK-8, n = 6). At 24:00, animals and food were weighed and a sample of blood was obtained from the caudal vein.
In assay 3, twenty rats were fasted from 09:00 until 18:00 and then fed ad libitum with pellets (cookies) prepared from ground standard chow, flaxseed oil and egg white (6/4/2). Rats were randomly assigned to receive either saline (n = 10) or L-365,260 (1 mg/kg; n = 10) at 21.30. Five animals of each group received either saline or CCK-8 (10 μg/kg) at 22:00. Animals were killed by decapitation, under inhalational anesthesia with isoflurane, at 24:00 and perirenal (Per-WAT), mesenteric (Mes-WAT) and subcutaneous (Sc-WAT) WAT were dissected and frozen in liquid nitrogen. Flaxseed oil was used because of its elevated content in linolenic (>50%) and linoleic acids (>15%), which are absent in the standard rodent chow, and therefore, useful as a probe to trace dietary TGs by 1H-NMR. Blood was collected for plasma biochemistry analysis. Per-WAT, Mes-WAT and Sc-WAT were collected for LIN quantification, gene expression analysis and LPL activity measurement. Both doses and timing for drug administration, as well as sampling and the sacrifice of animals were scheduled on the basis of previous studies by our group (Ruiz‐Gayo et al. 2000, Cano et al. 2008, Merino et al. 2008a ) showing that the efficacy of effects of CCK-8 on energy metabolism was optimal when administered during the initial phase of the dark period of the circadian cycle.
Experiment 2: Chronic treatment with CCK-8
Chronic treatment with CCK-8 was carried out in order to explore an eventual loss of efficacy of CCK-8. Another group of 14-week-old male Sprague-Dawley rats was submitted to chronic treatment (i.p., twice daily, at 09:00 and 18:00) with either saline (n = 6) or CCK-8 (n = 6, 10 μg/kg) and maintained on standard rodent chow. On day 12 of treatment, animals were administered their corresponding treatment at 09:00 and fasted until 18:00. They were then given free access to the flaxseed oil pellets described earlier. Rats received saline or CCK-8 at 18:00 and were decapitated at 24:00.
Plasma biochemistry and tissue lipid quantification
Glucose (GTM, Roche), triglycerides (Biolabo, Maizy, France) and non-esterified free fatty acids (NEFA; Wako Bioproducts) were measured by colorimetric methods. Plasma leptin, insulin and CCK levels were analyzed by EIA (Phoenix Pharmaceuticals, Karlsruhe, Germany, for leptin and CCK, and Mercodia, Uppsala, Sweden, for insulin). Lipids were extracted from WAT and liver in chloroform/methanol (2/1) following the method of Folch with modifications (Herrera & Ayanz 1972).
Western blotting of ANGPTL-4
Plasma samples (7–10 µg proteins) were loaded in Laemmli buffer and size separated by SDS-PAGE. After protein transfer to PVDF membranes (GE Healthcare) and blocking with 5% nonfat dried milk/Tween-PBS (1 h), a commercially available rabbit polyclonal antibody validated for Western blotting and directed against the N-terminal domain of ANGPTL-4 (1:1000; Abcam ab2920), which is the domain interacting with LPL (Sukonina et al. 2006), was applied overnight at 4°C. After washing, an IgG-peroxidase-conjugated secondary antibody was applied for 1 h (1:5000). Blots were incubated in chemiluminescence reagents (ECL Prime, GE Healthcare) and bands were detected using the ChemiDoc XRS+ Imaging System (BioRad). ANGPTL-4 values were normalized to serum albumin levels (Ponceau S Red; Sigma). Values are expressed as percent of the saline group.
Lipoprotein lipase activity assay
Fifty milligrams of WAT were homogenized in 0.8 mL Tris–HCl (0.2 mol/L, pH = 8.2). After centrifugation (10 min, 1250 g ), the aqueous phase was transferred to Eppendorf tubes containing 50 µL preheated fetal calf serum (60°C; 10 min). Proteins were precipitated with cold acetone (−20°C), and then washed with ether and dried. The resulting powder was re-suspended in 1 mL 0.05 mol/L NH4OH/NH4Cl buffer (pH = 8.1). Samples were assayed for LPL activity as previously described (Sugden et al. 1993). Briefly, 0.2 mL samples were incubated (30 min, 37°C) in a buffer prepared from 0.05 mL water, 0.04 mL 0.2 mol/L Tris–HCl buffer (pH = 8.2; 0.15 mol/L NaCl, 6% BSA), 0.02 mL preheated fetal calf serum and 0.04 mL C14-labeled triolein (Perkin-Elmer) solution (69 mg cold triolein, 3.3 mg lecithin and 462 kBq 14C-triolein, and 5 mL glycerol). The enzymatic reaction was stopped by the addition of 3 mL chloroform/heptane/methanol (1/1/1), and free 14C-oleic acid extracted with 1 mL 0.1 M KBO3/K2CO3 (pH = 8.5) and quantified. All samples were run in triplicate. Values are expressed in fkat/mg prot.
Evaluation of dietary triglyceride uptake by 1H-nuclear magnetic resonance
WAT samples were processed for total lipid extraction with chloroform/methanol. Ten milligrams of the extract were dissolved in deuterated chloroform and 1H-NMR spectra recorded at 400 MHz in a Bruker AdvanceIII apparatus (Bruker, Ettlingen, Germany), at room temperature. Total LIN content was estimated by integrating signals corresponding to methylenic protons located in positions 11 and 14 (2.77 ppm; LIN11,14), which are unique in LIN as they have two neighboring double bonds. Integration values were referred to signal intensities of the α-protons (2.31 ppm), which are common to all fatty acids (Knothe & Kenar 2004). This ratio (LIN11,14/α) allows the comparison of the amount of LIN between controls and CCK-8-treated animals. Although 1H-RMN has not been generally used for quantification of complex mixtures due to signal overlapping, it allows the simultaneous determination and quantification of multiple analytes in biological samples (Pauli et al. 2012) and has been validated for TG quantification in rat adipose tissue (Mosconi et al. 2011).
Cholecystokinin receptor gene silencing
Differentiated pre-adipocytes (Supplementary Data 1 and Supplementary Fig. 2, see section on supplementary data given at the end of this article) were transfected with siRNAs for Cckar and Cckbr (s234619 and s218025, respectively; Life Technologies) in 24-well plates. Cckar and Cckbr are the genes encoding for CCK-1R and CCK-2R proteins, respectively. Briefly, for each well, siRNAs (40 nmol/L) were diluted in 50 μL Opti-MEM (Life Technologies), incubated 15 min (25°C) and then added to a solution containing 3 μL lipofectamine RNAiMAX (Life Technologies) diluted in 50 μL Opti-MEM. After 15 additional min (25°C), 100 μL of the mixture were added dropwise to wells containing 400 μL Opti-MEM. The medium was replaced by differentiation media after overnight incubation. Two days after transfection, the medium was changed to α-MEM containing 0.1% BSA (Lee et al. 2014), then CCK-8 (10−6 mol/L) was added and incubated for 2 h (37°C, 5% CO2). Media was removed and mRNA (2 wells) was extracted as detailed below. Assays were replicated three times. Positive controls for transfection were performed by using Block-iT Alexa Fluor Red (Life Technologies). A Silencer Select Negative Control (4390843, Thermo Fisher Scientific) was used. All samples were run in pentaplicate.
Quantitative real-time PCR
Total RNA was extracted by using the Tri-Reagent protocol (Sigma). cDNA was then synthesized from 1 µg total RNA by using a high-capacity cDNA RT kit (Applied Biosystems). Quantitative RT-PCR was performed by using designed primer pairs (Integrated DNA Technologies, USA) for Angptl-4 (forward 5′-TCCGAGGGGACCTTAACTGT-3′; reverse 3′-CCGTTGCCGTGG AATAGAGT-5′), Cckar (forward 3′-GTTGCCAAGTGACGCTATGC-3′, reverse 5′-CCACCACCATCACAATCCCA-3′), Cckbr (forward 3′-CGGGTGTCTCCCTTCTCAAC-5′, reverse 5′-CGTTTCCGCCAACACTCATC-3′) and Lpl (forward 5′-TCTAACTGCCACTTCAACCACAGCAG-3′, reverse 5′-CAAGACTGTACCCTAAGAGGTGGAC-3′). SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) was used for amplification according to the manufacturer’s protocols in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Values were normalized to the housekeeping gene actin (Rn00667869_m1) and 18S ribosomal RNA (Rn03928990_g1). The ∆∆C(T) method was used to determine relative expression levels. Statistics were performed using ∆∆C(T) values (Livak & Schmittgen 2001). All samples were run in duplicate.
Statistics
Individual effects within a given group were analyzed by using a one-way ANOVA, followed by Newman–Keuls’ post hoc test. Correlations were analyzed by calculating Pearson correlation coefficients. Statistical significance was set at P < 0.05. All statistics were performed using Graph Pad Prism 7 (GraphPad Software).
Results
Acute treatment with CCK-8 reduced plasma ANGPTL-4 levels
As illustrated in Fig. 2A and B, CCK-8 (10 µg/kg) inhibited food intake (P < 0.05) and reduced BW (P < 0.05). Moreover, CCK-8 reduced circulating ANGPTL-4 levels (Fig. 2C; P < 0.05), an effect that cannot be attributed to the inhibition of food intake triggered by CCK-8 as pair-feeding to 10 μg/kg CCK-8-treated rats (Fig. 2A and B) did not modify plasma ANGPTL-4 (Fig. 2C). The CCK-1R antagonist SR-27,897 blocked the effect of CCK-8 on food intake (Fig. 2D; P < 0.05) and BW (Fig. 2E; P < 0.05). These effects were also antagonized by the CCK-2R antagonist, L-365,260. In contrast, the effect of CCK-8 on ANGPTL-4 levels was selectively blocked by L-365,260 (Fig. 2F; P < 0.01). Circulating levels of glucose, TG, NEFA, leptin, insulin and CCK were unaffected by CCK-8 (Table 1).
Effect of acute treatment with CCK-8 and CCK receptor antagonists on food intake, body weight and plasma ANGPTL-4. Effect of CCK-8 on food intake (A), body weight (B) and ANGPTL-4 plasma levels (C). Pair fed rats received pre-weighed food equivalent to food-intake observed in rats receiving 10 μg/kg CCK-8. Panels (D), (E) and (F) illustrate the effect of SR-27,897 (CCK-1 receptor antagonist) and L-365,260 (CCK-2 receptor antagonist) on the decrease in food intake, body weight and plasma ANGPTL-4 triggered by 10 μg/kg CCK-8, respectively. Values are means ± s.e.m. (n = 6). *P < 0.05; ***P < 0.001, compared to the control saline group (Newman–Keuls’ test).
Citation: Journal of Endocrinology 236, 3; 10.1530/JOE-17-0580
Effect of acute and chronic treatment with CCK-8 on plasma parameters.
| Acute treatment | Chronic treatment | |||
|---|---|---|---|---|
| Saline | CCK-8 (10 µg/kg) | Saline | CCK-8 (10 µg/kg) | |
| Glucose (mg/dL) | 110.5 ± 3.4 | 110.8 ± 2.3 | 101.9 ± 4.0 | 90.9 ± 2.6 |
| TG (mg/dL) | 159.6 ± 25.7 | 116.6 ± 20.8 | 152.6 ± 12.7 | 114.4 ± 10.3 |
| NEFA (mg/dL) | 43.4 ± 5.1 | 35.3 ± 3.4 | 37.8 ± 2.4 | 31.9 ± 1.2* |
| Insulin (ng/mL) | 0.77 ± 0.13 | 0.83 ± 0.18 | 1.85 ± 0.24 | 0.92 ± 0.14* |
| Leptin (ng/mL) | 2.3 ± 0.5 | 4.1 ± 1.2 | 0.87 ± 0.1 | 0.26 ± 0.1* |
| CCK (ng/mL) | 0.77 ± 0.10 | 0.74 ± 0.09 | 0.47 ± 0.01 | 0.53 ± 0.06 |
Acute treatment with a single dose of CCK-8 (10 μg/kg) had no effect on plasma levels of glucose, triglycerides (TG), non-esterified fatty acids (NEFA), insulin, leptin and total cholecystokinin immunoreactivity (CCK). Chronic CCK-8 treatment had no effect on plasma glucose, NEFA or CCK, but reduced TG, insulin and leptin levels. Values are means ± s.e.m. (n = 6).
* P < 0.05 compared to the control group (Newman–Keuls’ test).
Acute treatment with CCK-8 repressed Angptl-4 expression and simultaneously enhanced LPL activity and TG content in subcutaneous and visceral WAT
WAT deposits were weighed and compared between the different groups of animals. CCK-8 did not significantly modify the amount of WAT deposits (Fig. 3A). However, LPL activity (Fig. 3B), but not Lpl gene expression (Fig. 3C), was significantly enhanced by CCK-8 in both Sc-WAT and Vis-WAT (P < 0.05 in all tissues). In addition, CCK-8 reduced Angptl-4 mRNA levels in these WAT deposits (Fig. 3D; P < 0.05).
Effect of acute treatment with CCK-8 and the CCK-2R antagonist, L-365,260, on adiposity, LPL activity, Angptl-4 expression and TAG uptake in subcutaneous and visceral adipose WAT. (A) Effect of 10 μg/kg CCK-8 and L-365,260 on the amount of subcutaneous, perirenal and mesenteric WAT. (B) LPL activity, but not Lpl expression (C), was increased by CCK-8 in all WAT deposits analyzed and its effect was antagonized by L-365,260. (D) Angptl-4 expression was repressed by CCK-8 in all WAT deposits. L-365,260 prevented the effect of CCK-8. Angptl-4 values were normalized to the housekeeping gene β-actin and 18S ribosomal RNA. (E) Typical 1H-NMR records corresponding to a total lipid extract of WAT from control (black line) and CCK-8-treated rats (grey line) that consumed a linolenic/linoleic-enriched diet previously to treatment with 10 μg/kg CCK-8. CCK-8 enhanced the amount of linolenic/linoleic acid in the subcutaneous (F), perirenal (G), and mesenteric WAT (H) and its effect was prevented by L-365,260. Correlations between LIN11,14/α and LPL activity (I, J and K). Values are means ± s.e.m. (n = 5). *P < 0.05; **P < 0.01 and ***P < 0.001, compared to the control group (Newman Keuls’ test).
Citation: Journal of Endocrinology 236, 3; 10.1530/JOE-17-0580
Figure 3E shows a typical 1H-NMR spectrum of total lipids obtained from Per-WAT samples of rats that ate a linolenic/linoleic-rich (LIN) diet and that were treated with either saline (black line) or CCK-8 (grey line). Signals corresponding to methylene protons 11 and 14 from LIN, which appear as a triplet at 2.77 ppm, were integrated and normalized to the integral of the multiplet corresponding to the α-protons at 2.31 ppm, which is common to all fatty acids. This was used to estimate the relative proportion of LIN in each sample and evaluate the influence of CCK-8 on total LIN uptake. As illustrated in Fig. 3F, G and H, CCK-8 enhanced the ratio LIN11,14/α in all Sc-, Per- and Mes-WAT deposits (P < 0.05), and this effect was abolished by L-365,260. Positive correlations between LIN11,14/α and LPL activity were identified in Sc-WAT (Fig. 3I; P < 0.05), Per-WAT (Fig. 3J; P < 0.001) and Mes-WAT (Fig. 3K; P < 0.01).
The effect of CCK-8 on Angptl-4 expression was abolished by silencing Cckbr expression in adipocyte precursor cells
Because in vivo assays strongly suggested that CCK-2R is involved in regulating Angptl-4 expression in adipose tissue, an in vitro experiment carried out in pre-adipocytes was designed to confirm this hypothesis. Pre-adipocytes were differentiated from the subcutaneous and visceral WAT-derived stromal vascular fractions (Supplementary Data 1 and Supplementary Fig. 2) and transfected with siRNAs for Cckar (corresponding to CCK-1R protein) or Cckbr (corresponding to CCK-2R protein). The effectiveness of gene knockdown on Cckar and Cckbr mRNA levels is illustrated in panels 4A and 4B (Cckar) and 4C and 4D (Cckbr), respectively.
Figure 4E and F show the effect of CCK-8 on Angptl-4 expression in visceral and subcutaneous pre-adipocytes after Cckar and Cckbr mRNA silencing. CCK-8 repressed Angptl-4 expression, and this effect was specifically abolished in cells carrying the silenced Cckbr gene (P < 0.05). CCK-8 had no effect on Lpl gene expression (Fig. 4G and H).
Effect of acute treatment with CCK-8 on Angptl-4 expression in visceral and subcutaneous pre-adipocytes. A and B and C and D panels illustrate the efficacy of the silencing procedure in Cckar and Cckbr expression, respectively. CCK-8 (10−6 mmol/L) inhibited Angptl-4 expression in pre-adipocytes obtained from perirenal (E) and subcutaneous WAT (F). CCK-8 had no effect on Lpl expression in both types of pre-adipocytes (G and H). Angptl-4 and Lpl values were normalized to the housekeeping gene 18S ribosomal RNA and β-actin. Cckbr, but not Cckar, expression silencing fully prevented the effect of CCK-8 in both pre-adipocyte populations. Values are means ± s.e.m. (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group (Newman Keuls’ test).
Citation: Journal of Endocrinology 236, 3; 10.1530/JOE-17-0580
Chronic treatment with CCK-8 also reduced Angptl-4 expression and LPL activity in adipose tissue
Figure 5 illustrates the effect of chronic CCK-8 treatment on the same parameters analyzed in the previous study. Under these conditions, chronic CCK-8 limited BW gain (5.20 ± 2.04 g in the CCK-treated group vs 11.00 ± 2.45 g in the saline group). The last injection of CCK-8 caused a decrease in BW (−1.40 ± 0.30 g in CCK-8-treated vs 0.70 ± 0.20 g in saline-treated rats; P < 0.05), measured 6 h after the last CCK-8 administration. Circulating levels of ANGPTL-4 were reduced by chronic CCK-8 (Fig. 5A; P < 0.01). In addition, a robust decrease of Angptl-4 mRNA expression (Fig. 5B; **P < 0.01, ***P < 0.001), together with an enhancement of LPL activity (Fig. 5C; *P < 0.05, **P < 0.01), was detected in both visceral and subcutaneous adipose tissues of rats chronically treated with CCK-8. Chronic CCK-8 had no effect on Lpl expression (Fig. 5D).
Effect of chronic CCK-8 on Angptl-4 expression, LPL activity, adiposity, triglyceride uptake in subcutaneous and visceral adipose tissues and plasma biochemistry. Chronic CCK-8 decreased circulating ANGPTL-4 (A), repressed Angptl-4 expression in subcutaneous and visceral WAT (B), and enhanced LPL activity (C). Lpl expression was not modified by chronic CCK-8 (D). Angptl-4 values were normalized to the housekeeping gene β-actin and 18S ribosomal RNA. The total amount of adipose tissues was not modified by chronic CCK-8 (E), but the uptake of linolenic/linoleic acid was enhanced in all WAT deposits (F, G and H). Correlations between LIN11,14/α and LPL activity (I, J and K). Values are means ± s.e.m. (n = 6). *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the control group (Newman–Keuls’ test).
Citation: Journal of Endocrinology 236, 3; 10.1530/JOE-17-0580
Although the weights of the adipose depots were not modified by chronic CCK-8 (Fig. 5E), LIN uptake was significantly increased by CCK-8 in Sc-WAT and Vis-WAT (Fig. 5F, G and H; P < 0.05). As observed in the case of acute CCK-8 treatment, a positive linear correlation between LIN11,14/α and LPL activity was detected in Sc-WAT (Fig. 5I; P < 0.001), Per-WAT (Fig. 5J; P < 0.01) and Mes-WAT (Fig. 5K; P < 0.05). Chronic CCK-8 treatment had no effect on plasma glucose, NEFA or CCK, but reduced TG, leptin and insulin levels (Table 1).
Chronic CCK-8 treatment had no effect on food intake during the treatment (Fig. 4 and supplementary data).
Discussion
This study reports the first evidence of CCK-8-regulated hydrolysis of TG and increased uptake of FAs by WAT and supports a new role for CCK in connecting the gut and WAT to drive TG homeostasis. We demonstrate that CCK-8, by acting on adipocyte CCK-2R, reduces WAT Angptl-4 gene expression as well as circulating ANGPTL-4 levels, leading to the activation of WAT LPL (Sukonina et al. 2006). This mechanism suggests the existence of an axis connecting CCK-2R, ANGPTL-4 and LPL (Fig. 6) that would drive the incorporation of dietary fat into WAT and would be involved in the expandability of WAT deposits during postprandial periods.
Proposed model of regulation of the lipoprotein lipase (LPL)–angiopoietin-like protein-4 (ANGPTL-4) axis by CCK-8. (1) During fasting, CCK-2 binding sites are poorly occupied as plasma levels of CCK remain low. Under these conditions signaling pathways downstream CCK-2R (for instance, PI3K/Akt signaling pathway) display low activity and the Angptl-4 gene is transcribed. Therefore plasma levels of ANGPTL-4 are elevated, and LPL remains inactive (low activity). (2) In postprandial periods, increased levels of CCK and further occupation of CCK-2R promote the activation of PI3K/Akt, and repress the expression of Angptl-4, by acting on CCK-2R in adipocytes. As a consequence, plasma ANGPTL-4 levels decrease and LPL become active, allowing the uptake of fatty acids by WAT. CM, chylomicrons; TAG, triglycerides.
Citation: Journal of Endocrinology 236, 3; 10.1530/JOE-17-0580
The most important finding of our study is the downregulation of Angptl-4 mRNA levels together with the increase in LPL activity in Sc and visceral WAT evoked by CCK-8, which occurs concomitantly with the reduction of circulating ANGPTL-4. The activation of LPL by CCK-8 is not linked to the increase of adipose Lpl gene expression, since mRNA Lpl mRNA levels were not modified by CCK-8. Likewise, the lack of effect on plasma ANGPTL-4 levels observed in pair-fed rats allows us to discard the influence of the decrease of food intake triggered by CCK-8 on Angptl-4 gene expression/LPL activity. In regards to the CCK receptor subtype involved in CCK-8 effects, our data demonstrate the specific involvement of CCK-2R. Importantly, the effect of CCK-8 was not diminished after chronic exposition to this hormone, suggesting that CCK-2R-mediated effects are apparently not submitted to desensitization mechanisms. This result would be coherent with a lack of tolerance to CCK-2R-mediated effects and might have important implications for eventual therapeutic uses of CCK analogues.
The downregulation of adipose Angptl-4 gene expression is coherent with the decrease of plasma ANGPTL-4 concentration, as WAT is one of the main sources of circulating ANGPTL-4 (Yoon et al. 2000, Bergö et al. 2002) and suggest an autocrine/paracrine effect of ANGPTL-4 on LPL activity in this tissue. This possibility is supported by the lack of effect of CCK-8 on both Angptl-4 and Lpl mRNA levels within the liver (Fig. 3 and supplementary data), which is the other relevant origin of ANGPTL-4 in rodents. The finding that CCK-8 downregulates Angptl-4 gene expression in pre-adipocytes, together with the specific blockage of this response in cells when the CCK-2R gene is silenced, further demonstrates the involvement of adipocyte CCK-2R on CCK-8 effects and gives support to the concept that CCK systems within WAT play a role on the regulation of LPL in WAT. Nevertheless, although our data strongly suggest that an increase in LPL activity reflects the activation of endothelial LPL, the activation of non-endothelial pools of LPL cannot be excluded.
Our current data show that the adipotrophic effect of CCK-8 might be endowed with physiological relevance for lipid and energy homeostasis and be complementary to insulin’s actions in regulating TG storage (Czech et al. 2013). It should be noted that a possible CCK-8-mediated increase in intestinal absorption of TG could theoretically account for CCK-8’s effects. However, this possibility can be ruled out as the highest dose used in the study (10 μg/kg) did not increase the absorption rate constant (ka) or the bioavailability of orally administered TGs (Fig. 1 and supplementary data). In addition to this, the lack of effect of acute CCK-8 on plasma TG, together with the decrease detected after chronic treatment, support the concept that CCK-8’s effects on WAT are not linked to a rise in plasma TG. In any case, the effect of CCK-8 does not appear to be associated to an increase in plasma insulin levels, as CCK-8 failed to modify plasma insulin levels and even triggered a significant decrease of the hormone in chronically treated animals. These data are in apparent contradiction with the role of CCK as an insulin secretagogue (Ahrén & Lundquist 1981, Verspohl et al. 1986, Rossetti et al. 1987, Lo et al. 2011), and particularly with the study by Ahrén et al. (2000), reporting an increase in plasma insulin after CCK infusion (Ahrén et al. 2000). Our results could be due to the different experimental conditions. Thus, Ahrén and coworkers administered CCK-8 at a constant flow during 90 min to humans that adhered to a similar food intake protocol. In our study, an i.p. bolus of CCK was administered to free-feeding rats. In accordance with the satiating effect of CCK, animals receiving CCK-8 displayed a significant decrease in food intake, that taken together with the inhibitory effect of CCK-8 on gastric emptying (Fried et al. 1991), could balance the insulin secretagogue effect of CCK-8. In regard to the different basal insulin levels observed in rats submitted to the acute/chronic treatment, it can be argued that differences in the experimental protocol may explain why chronically treated rats (that received an i.p. injection of saline 6 h before sacrifice) displayed higher insulin values than acutely treated rats (submitted to the same protocol, but 2 h before sacrifice). In fact, stress has been shown to inhibit insulin release (Begg & Woods 2013, Kuo et al. 2015). Therefore, targeting CCK pathways might be a useful strategy to improve hypertriglyceridemia (HTG) in diabetes. This is relevant because HTG is a main metabolic disorder integral to the cluster of diabetes-associated cardiovascular risks that remains partially unresolved (Chait & Goldberg 2017). In any case, further research focusing on the effect of CCK-8 on HTG management in experimental models of diabetes is necessary to assess such a possibility.
The effect of CCK-8 was not due to an increase of plasma leptin as acute CCK-8 did not significantly modify leptin levels. As observed in the case of insulin, chronic CCK-8 treatment also decreased plasma leptin, a finding that could be linked to the inhibitory effect of CCK-8 on BW. An important question that emerges from these data concerns the adequacy of the dose-range of CCK-8 used in our study. Most of the experiments have been carried out with the dose of 10 µg/kg, a dose that apparently yields plasma CCK concentrations within the physiological range (Cano et al. 2008). In accordance, plasma CCK levels were not modified by CCK-8 in our study. It should be noted that the relatively rapid degradation of CCK (Koulischer et al. 1982) could mask the influence of exogenous CCK-8 on total CCK levels. In addition, commercial CCK antibodies used for CCK quantification recognize a mixture of molecular forms derived from pre-proCCK that are present in blood (Reeve et al. 1986), making proper quantification of plasma bioactive CCK fragments difficult.
The effect of acute CCK-8 on Angptl-4 gene expression and LPL activity is endowed with functional consequences as CCK-8-treated animals that consumed linolenic/linoleic acid (LIN)-enriched fat incorporated LIN to WAT more efficiently than their saline-treated counterparts. This finding is supported by the positive correlation between LPL activity and the ability of the different adipose tissues analyzed to incorporate LIN. Interestingly, the effect of chronic CCK-8 on Angptl-4 mRNA levels, LPL activity and LIN uptake was similar to that observed after acute treatment with the hormone, suggesting the lack of tolerance to this CCK effect. Finally, the CCK-2R antagonist, L-365,260, specifically blocked the effect of CCK-8 on LIN uptake giving further support to the involvement of CCK-2Rs in dietary TGs management.
Taken together, the results of the current study support the concept of a CCK-2R/ANGPTL-4/LPL axis that regulates postprandial uptake of TG into WAT (Fig. 6) and completes the mechanisms proposed by Dijk & Kersten (Dijk & Kersten 2014) concerning the relevance of an ANGPTL-4/LPL axis aimed at clearing TGs from both chylomicrons and VLDL proteins. In regard to the mechanism involved in CCK-8-mediated repression of Angptl-4 expression and decrease of plasma ANGPTL-4 levels, a plausible possibility would involve the PI3K/Akt pathway as this signalling pathway, which has been shown to be pivotal for insulin-mediated repression of the Angptl-4 gene (Kuo et al. 2014), is activated by CCK-2R (Dufresne et al. 2006). In any case, the versatility of downstream signaling pathways coupled to CCK-2R (Dufresne et al. 2006) allows one to hypothesize the involvement of other signaling pathways, such as PPAR-γ (Burns & Heuvel 2007), that is also known to govern Angptl-4 expression.
It is important to highlight that ANGPTL-4, in addition to its role in regulating TG uptake, has been shown to govern angiogenesis in adipose tissue (Hato et al. 2008). Although data in the literature are controversial, and some studies have demonstrated an anti-angiogenic effect of the C-terminal domain of ANGPTL-4 (Yang et al. 2008), most studies have identified ANGPTL-4 as a potent pro-angiogenic and anti-inflammatory agent that favors the expandability of WAT (Kadomatsu et al. 2011). In this context, the potential use of CCK-related drugs to improve WAT functionality in insulin-resistant conditions might be limited by an eventual inhibition of angiogenesis. Moreover, the lack of data in regard to the effect of CCK-8 on human WAT limits the impact of our study in order to design pharmacotherapeutic strategies based on the use of CCK analogues as tools for insulin resistance management. In any case, the possibility that the system conformed by CCK receptors, LPL and ANGPTL-4 could be an important target for improving metabolism deserves further attention and should be validated in human WAT.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-17-0580.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this research reported.
Funding
Ministerio de Economía y Competitividad (BFU2016-78556R, BFU2014-51836-C2-2-R; BFU 2012-35353; CTQ2015-64624-R), European Regional Development Fund, Universidad San Pablo-CEU and CIBEROBN. F H-N and D D-R are supported by the postgraduate fellowship program of Universidad San Pablo-CEU. A P is a postgraduate fellow of Ministerio de Economía y Competitividad (BES-2012-063773).
Author contribution statement
A P, V C, G D, J P-C and B M contributed to data acquisition and interpretation. B M, C S, J A C and M R-G designed the study and wrote the manuscript. J P-C, M F-A, C S, J A C and M R-G drafted and revised the manuscript. All authors approved the final version of the manuscript.
Acknowledgements
The authors would like to thank I Isla-Gayo for Fig. 6 design, and S Álvarez for helpful technical assistance.
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