Background
Obesity has reached epidemic proportions globally, and more than one-third of adults (35.7%) in USA are obese. Being overweight or obese increases the risk of diseases including cancer, coronary heart disease, type II diabetes, hypertension and stroke. Compelling evidence has demonstrated that dietary fat intake is a major cause of obesity, diabetes, and the associated metabolic syndromes [
1,
2].
Human apoA-IV is a 46-kDa protein produced by enterocytes of the small intestine and its production is stimulated by fat absorption [
3]. ApoA-IV is secreted in association with triacylglycerol (TG) rich chylomicron particles. After entering the circulation, apoA-IV rapidly dissociates from chylomicrons and then transfers into high-density lipoproteins or becomes a lipoprotein-free fraction of the plasma in the postprandial state [
4]. ApoA-IV has a number of well-established functions, including the inhibition of food intake, anti-inflammatory role, reverse cholesterol transport, and the regulation of glucose metabolism through the stimulation of insulin secretion and inhibition of hepatic gluconeogenesis [
5-
9]. Although the production of apoA-IV is stimulated by fat absorption, chronic ingestion of high fat (HF) feeding caused the adaptation of the intestinal apoA-IV response to lipid feeding. Weinberg et al. [
10] first reported the adaptation of plasma apoA-IV in response to chronic feeding of a HF diet in humans. They observed that consumption of an HF diet for 1 week significantly elevated plasma apoA-IV, but after two weeks on the HF diet, the apoA-IV level returned back to baseline. Kalogeris and Painter [
11] found that plasma apoA-IV increased initially by 40% in response to intra-gastric administration of fat emulsion within the first one to two days, but this was followed by the diminished responses with no increase in plasma apoA-IV levels by four days of fat feeding. Similarly, the jejunal mRNA levels and mucosal apoA-IV protein synthesis also showed time-dependent refractoriness to fat administration, suggesting both posttranslational (protein clearance) and/or pre-translation (transcriptional) adaptation of the intestinal apoA-IV production.
It is well established that circulating pro-inflammatory cytokines [e.g. tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6)] are induced in obesity and these pro-inflammatory cytokines play a crucial role in the development of metabolic syndrome [
12-
14]. Ding et al. reported that HF diet feeding and bacteria interaction promoted TNF-α mRNA production and intestinal inflammation in mice; and the increase TNF-α preceded obesity was strongly and significantly associated with progression of obesity and development of insulin resistance [
15]. Recently, Ji et al. showed that intra-duodenal infusion of fat emulsion caused mucosal mast cells activation and increased lymphatic secretion of pro-inflammatory cytokines IL-6 [
16]. These observations suggest that pro-inflammatory cytokines derived from gut may contribute to the development of obesity and associated metabolic syndrome.
It was reported that treatment with cytokines TNF-α or IL-6 in cultured pig hepatocytes decreased apoA-IV mRNA levels [
17], suggesting that the cytokines may contribute to dietary-induced obesity through down-regulation of the important satiety signal apoA-IV expression [
18]. To determine whether pro-inflammatory cytokines are involved in the attenuated response of apoA-IV to chronic HF feeding, we performed
in vitro study to investigate the effect of human recombinant cytokines TNF-α, IL-6 or their combination on apoA-IV expression in response to the treatment of Linoleic acid (LA, an important dietary fatty acid) in cultured human intestinal Caco2 cells. Our results suggest that pro-inflammatory cytokines inhibit apoA-IV production induced by LA in Caco2 cells.
Methods
Materials
Linoleic acid, sodium taurocholate (TC) and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human IL-6 (r-h-IL-6) and recombinant human TNF-α (r-h-TNF-α) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Dulbecco’s modified essential medium (DMEM), fetal bovine serum (FBS) and antibiotic: antimycotic mixture were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA).
Cell cultures
Caco-2 cells, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), were grown in DMEM containing high glucose, 20% FBS, and a 1% antibiotic: antimycotic mixture. 70-80% confluent Caco-2 cells were plated onto 24-well plastic dishes (Becton-Dickinson Labware, Lincoln Park, NJ, USA) at initial densities of 1 × 10
5 cells/well in complete growth medium. The medium was changed every other day for 14–21 days. This procedure is known to induce differentiation of Caco-2 cells into more enterocyte-like cells [
19].
LA and cytokine treatment to Caco2 cells
To prepare LA stocks (20 ×), 20 mM LA and 10 mM TC were mixed and stored at −20°C. To prepare TNF-α and IL-6 (1000 ×, 10 mg/ml) stocks, r-h-IL-6 and r-h-TNF-α were dissolved in phosphate buffered saline (PBS). The differentiated Caco2 cells were incubated in growth medium with or without 20 ng/ml TNF-α, or 20 ng/ml IL-6, respectively, or a mixture of the both (20 ng/ml/each) for the indicated time, and then changed into DMEM/ high glucose/1% FBS with or without indicated amount of LA: TC for the time indicated, with 0.5 mM TC as vehicle control. The cells were harvested and lysed, and the total RNA was isolated.
Real-time RT-PCR
Total RNA was isolated from cells with RNeasy Mini Kit (Qiagen, Germantown, MD, USA). First-strand cDNA was synthesized from 1 μg total RNA with Scripts™ cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer’s instruction. Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories Inc. Hercules, CA, USA) with an iCycler iQ Detection System (icycler iQ. Multicolor Real-time PCR Detection System, Bio-Rad) and normalized to β-actin. All primers were purchased from Integrated DNA Technologies (Coralville, IA,USA). PCR primer pairs used were: human apoA-IV forward, 5′-ACCCAGCTAAGCAACAATGC-3′, and reverse, 5′-TGTCCTGGAAGAGGGTACTGA-3′; human Il6 forward, 5′-TGATGGATGCTACCAAACTGG-3′, and reverse, 5′-TTCATGTACTCCAGGTAGCTATGG-3′; human Tnfα forward, 5′-TCTTCTCATT CCTGCTTGTGG-3′, and reverse, 5′- GGTCTGGGCCATAGAACTGA-3′; human apoC-III forward, 5′-AGACCGCCAAGGATGCACTGA-3′, and reverse, 5′-TCTGACCTCAGGGTCCAAATCC-3′; βActin forward, 5′-TTGCTGACAGGAT-GCAGAAGGAGA-3′, and reverse, 5′-TCAGTAACAGTCCGCCTAGAAGCA-3′.
Enzyme linked immunosorbent assay (ELISA)
ELISA was performed using Human ApoA-IV ELISA Kit from Millipore Corporation (Billerica, MA, USA), Human IL-6 and Human TNF-α ELISA Kits from RayBiotech (Norcross, GA, USA) according to the protocol provided by the manufacturer. The culture mediums collected from the cells treated with LA with or without cytokines were used to measure the proteins released from the cells by ELISA.
Statistics
Data represent Mean ± SE from three or four wells in each experiment of at least two independent cell culture experiments. Significance of differences was determined by one-way ANOVA or two-way ANOVA followed by Tukey test method. P value less than 0.05 was considered significant.
Discussion
ApoA-IV is produced dominantly in the gut and has many physiological functions which include satiety, regulation of glucose metabolism, reverse cholesterol transport and also anti-inflammation [
6,
8,
9,
22]. Therefore, the reduced apoA-IV production in response to active lipid absorption after chronic HF diet-feeding has been implicated in the diet-induced obesity and metabolic disorders. The purpose of this study was to test the hypothesis that the inflammatory cytokines expressed and released during chronic HF feeding potentially play an important role in this reduced apoA-IV response to fat absorption. Linoleic acid (LA) is an important dietary fatty acid and a major component in many HF diets or the lipids that have been used to induce apoA-IV production [
11,
23].
Caco-2 cells, a human colon carcinoma cell line, have been widely used as a model to study intestinal epithelial cell metabolism, including lipids and lipoproteins [
19,
24-
27]. However, it has not been tested previously whether LA stimulates apoA-IV expression in those cells. With different dose of LA and varying incubation times with the differentiated Caco2 cells, for the first time, we found that
apoA-IV gene expression and protein secretion was significantly increased in response to LA stimulation. Notably, apoA-IV production was significantly enhanced with lower amount of LA (0.25 mM) stimulation, but high dose of LA (1 mM) failed to increase apoA-IV protein production, although
apoA-IV mRNA was significantly increased with this amount of LA from 12 h to 24 h. It was well-known that fat induces both apoA-IV synthesis and secretion from the gut, but up to now, its molecular mechanism is not clear. Our observations that apoA-IV mRNA level was increased with the increase of LA amount, but protein secretion of apoA-IV could not induced by a higher amount of LA, indicating that higher amount of LA may activate or induce other related factors, e.g. IL-6 and TNF-α, which can impact apoA-IV’s translation and the stability.
ApoA-IV is a member of a closely linked
apoA-I/C-III/A-IV gene cluster, a target for acute phase proteins. A coordinately regulation of
apoC-III and
apoA-IV expression by lipids has been previously described [
20]. However, it is also reported that the regulation of the members of this cluster by fat and inflammatory processes in the gut is not shared by all members of the cluster, e.g. neither the magnitude of response to the stimuli, nor the behavior of apoA-IV and apoC-III was similar [
17]. Our data showed no difference in
apoC-III gene expression in the cultured Caco2 cells treated with LA, IL-6 or the both together. In contrast, significant change in
apoA-IV gene expression was observed when the Caco2 cells were exposed to the same cytokines. These results indicate a loss of the coordinated response reported previously in the animals [
20], and suggest a complex interplay of transcription factors modulated by species-specific signaling pathways in response to a specific stimuli.
Our data also showed that LA not only induced
apoA-IV gene expression, but also stimulated TNF-α and IL-6 secretion in Caco2 cells. Circulating cytokines, such as TNF-α or IL-6, have been shown to be elevated in obese humans [
28,
29] and this could be reversed with weight loss [
30]. While the mechanisms underlying obesity-associated inflammation are not fully understood, a number of studies suggested that the inflammation may derive from the accumulation of activated macrophages within adipose tissue, liver, and the enlarged adipocytes in obese animals and humans [
14,
31,
32]. Recent studies [
15,
16] suggest that 1) the gastrointestinal tract is another and early source of inflammation associated with diet, 2) the onset of intestinal inflammation precedes diet-induced increases in body weight, fat mass, as well as insulin resistance, and 3) the degree of TNF-α induction strongly correlates with diet-induced increases in weight, adiposity, plasma glucose, and insulin. Our data that LA-induced elevation of both
TNF-α and
IL-6 mRNAs and of protein levels in Caco2 cells further support the concept that inflammatory cytokines released from the gut induced by fatty acid is the early source of inflammation associated with the development of obesity and/or metabolic disorders because TNF-α and IL-6 are widely used as early biomarkers of inflammation of insulin resistance and the obesity relative diseases.
It has been reported that TNF-α activates NF-κB and other inflammatory pathways [
33,
34]. As we described above, LA not only induced
apoA-IV gene expression but also stimulated the gene expression and secretion of the cytokines. In addition, consistent with previous studies, TNF-α and IL-6, especially TNF-α, are further stimuli in the expression of the cytokines in the Caco2 cells, and enhance the effect of LA on these cytokines.
It rapidly became clear that early proinflammatory effects of HF diet, such as elevated TNF-α, could serve as a trigger for subsequent inflammation or insulin resistance through activating NF-kB and other inflammatory pathways involved in the etiology of insulin resistance and Type 2 diabetes. In addition to regulating metabolic homeostasis and intracellular signaling pathways that are well-established metabolic functions, inflammatory cytokines can impact on release of gut hormones such as glucagon-like peptide 2 (GLP-2) [
15]. Our observations that these two cytokines reduced apoA-IV production, but not
apoA-IV gene expression, imply that these cytokines’ inhibitory effects on apoA-IV not only occur at transcriptional levels but also at translational or posttranslational levels.
Pro-inflammatory molecules released by intestines, adipose and liver enter the circulation to influence the expression of apoA-IV at the gut. Our data that exogenous cytokines decreased apoA-IV production induced by LA support our hypothesis that pro-inflammatory cytokines are involved in reducing apoA-IV responses to fat feeding. These results support the notion that cytokines induction by chronic fat feeding is responsible for the blunted apoA-IV production normally stimulated by active fat absorption.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XL carried out the main body of this work and prepared the manuscript. MX performed the experiments. ML and YJ participated in the preparation and revision of the manuscript. ZF participated in designing and cooperating. All authors have read and approved the final manuscript.