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Background: Cystic Fibrosis (CF) is a genetic disease in which the intestine exhibits oxidative and inflammatory markers. As mitochondria are the central source and the main target of reactive oxygen species, we hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) defect leads to the disruption of cellular lipid homeostasis, which contributes to mitochondrial dysfunction., Methods: Mitochondrial functions and lipid metabolism were investigated in Caco-2/15 cells with CFTR knockout (CFTR -/- ) engineered by the zinc finger nuclease technique. Experiments were performed under basal conditions and after the addition of the pro-oxidant iron-ascorbate (Fe/Asc) complex., Results: Mitochondria of intestinal cells with CFTR -/- , spontaneously showed an altered redox homeostasis characterised by a significant decrease in the expression of PPARα and nuclear factor like 2. Consistent with these observations, 8-oxoguanine-DNA glycosylase, responsible for repair of ROS-induced DNA lesion, was weakly expressed in CFTR -/- cells. Moreover, disturbed fatty acid β-oxidation process was evidenced by the reduced expression of CPT1 and acyl-CoA dehydrogenase long-chain in CFTR -/- cells. The decline of mitochondrial cytochrome c and B-cell lymphoma 2 expression pointing to magnified apoptosis. Mitochondrial respiration was also affected as demonstrated by the low expression of respiratory oxidative phosphorylation (OXPHOS) complexes and a high adenosine diphosphate/adenosine triphosphate ratio. In contrast, the FAS and ACC enzymes were markedly increased, thereby indicating lipogenesis stimulation. This was associated with an augmented secretion of lipids, lipoproteins and apolipoproteins in CFTR -/- cells. The addition of Fe/Asc worsened while butylated hydroxy toluene partially improved these processes., Conclusions: CFTR silencing results in lipid homeostasis disruption and mitochondrial dysfunction in intestinal epithelial cells. Further investigation is needed to elucidate the mechanisms underlying the marked abnormalities in response to CFTR deletion.

Background: Understanding the specific mechanisms of rare autosomal disorders has greatly expanded insights into the complex processes regulating intestinal fat transport. Sar1B GTPase is one of the critical proteins governing chylomicron secretion by the small intestine, and its mutations lead to chylomicron retention disease, despite the presence of Sar1A paralog., Objective: The central aim of this work is to examine the cause-effect relationship between Sar1B expression and chylomicron output and to determine whether Sar1B is obligatory for normal high-density lipoprotein biogenesis., Approach and Results: The SAR1B gene was totally silenced in Caco-2/15 cells using the zinc finger nuclease technique. SAR1B deletion resulted in significantly decreased secretion of triglycerides (≈40%), apolipoprotein B-48 (≈57%), and chylomicron (≈34.5%). The absence of expected chylomicron production collapse may be because of the compensatory SAR1A elevation observed in our experiments. Therefore, a double knockout of SAR1A and SAR1B was engineered in Caco-2/15 cells, which led to almost complete inhibition of triglycerides, apolipoprotein B-48, and chylomicron output. Further experiments with labeled cholesterol revealed the downregulation of high-density lipoprotein biogenesis in cells deficient in SAR1B or with the double knockout of the 2 SAR1 paralogs. Similarly, there was a fall in the movement of labeled cholesterol from cells to basolateral medium containing apolipoprotein A-I, thereby limiting newly synthesized high-density lipoprotein in genetically modified cells. The decreased cholesterol efflux was associated with impaired expression of ABCA1 (ATP-binding cassette subfamily A member 1)., Conclusions: These findings demonstrate that the deletion of the 2 SAR1 isoforms is required to fully eliminate the secretion of chylomicron in vitro. They also underscore the limited high-density lipoprotein production by the intestinal cells in response to SAR1 knockout., (© 2017 American Heart Association, Inc.)

In the intracellular secretory network, nascent proteins are shuttled from the endoplasmic reticulum to the Golgi by transport vesicles requiring Sar1b, a small GTPase. Mutations in this key enzyme impair intestinal lipid transport and cause chylomicron retention disease. The main aim of this study was to assess whether Sar1b overexpression under a hypercaloric diet accelerated lipid production and chylomicron (CM) secretion, thereby inducing cardiometabolic abnormalities. To this end, we generated transgenic mice overexpressing human Sar1b (Sar1b(+/+)) using pBROAD3-mcs that features the ubiquitous mouse ROSA26 promoter. In response to a high-fat diet (HFD), Sar1b(+/+) mice displayed significantly increased body weight and adiposity compared with Sar1b(+/+) mice under the same regimen or with wild-type (WT) mice exposed to chow diet or HFD. Furthermore, Sar1b(+/+) mice were prone to liver steatosis as revealed by significantly elevated hepatic triglycerides (TG) and cholesterol in comparison with WT animals. They also exhibited augmented levels of plasma TG along with alterations in fatty acid composition. Concomitantly, they showed susceptibility to develop insulin insensitivity and they responded abnormally to oral glucose tolerance test. Finally, Sar1b(+/+) mice that have been treated with Triton WR-1330 (to inhibit TG catabolism) and orotic acid (to block secretion of very low-density lipoprotein by the liver) responded more efficiently to fat meal tests as reflected by the rise in plasma TG and CM concentrations, indicating exaggerated intestinal fat absorption. These results suggest that Sar1b(+/+) under HFD can elicit cardiometabolic traits as revealed by incremental weight gain, fat deposition, dyslipidemia, hepatic steatosis, insulin insensitivity and intestinal fat absorption., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Gastrointestinal peptides are involved in modulating appetite. Other biological functions attributed to them include the regulation of lipid homeostasis. However, data concerning PYY remain fragmentary. The objectives of the study were: (i) To determine the effect of PYY on intestinal transport and synthesis of cholesterol, the biogenesis of apolipoproteins (apos) and assembly of lipoproteins and (ii) To analyze whether the effects of PYY are similar according to whether cells are exposed to PYY on apical or basolateral surface., Methodology/principal Findings: Caco-2/15 cells were incubated with PYY (1-36) administered either to the apical or basolateral medium, at concentrations of 50 or 200 nM for 24 hours. De novo synthesis of cholesterol, cholesterol uptake, and assembly of lipoproteins were evaluated through the incorporation of [(14)C]-acetate, [(14)C]-cholesterol, and [(14)C]-oleate, respectively. Biogenesis of apos (A-I, A-IV, E, B-48 and B-100) was examined by the incorporation of [(35)S]-methionine. The influence of PYY on protein and mRNA levels of many key mediators of lipid metabolism was analyzed by Western blot and PCR, respectively. Our results show that PYY influenced cholesterol metabolism in Caco-2/15 cells depending on the site of PYY delivery. Apical addition of PYY significantly lowered the incorporation of [(14)C]-cholesterol likely via the reduction of NPC1L1, stimulated intracellular cholesterol synthesis probably through an increase in SREBP-2 expression, whereas it concomitantly increased apo A-I synthesis and decreased LDL secretion. In contrast, basolateral PYY reduced the production of chylomicrons (CM) as well as the biogenesis of apos B-48 and B-100, while lowering the expression of the transcription factors RXRα and PPAR(α,β)., Conclusions/significance: PYY is capable of influencing cholesterol homeostasis in intestinal Caco-2/15 cells depending on the site delivery. Apical PYY was able to decrease cholesterol uptake via NPC1L1 downregulation, whereas basolateral PYY diminished CM output through the biogenesis decline of apos B-48 and B-100.

Although postprandial hypertriglyceridemia is a major contributing factor in the development of atherosclerosis, little information is available on the effect of insulin resistance and diabetes on intestinal fat transport. The aim of the present study was to examine intracellular events that govern lipid transport and apolipoprotein (apo) B-48-containing lipoprotein assembly in the small intestine of Psammomys obesus, a model of nutritionally induced insulin resistance and type 2 diabetes. Animals with normoglycemia/hyperinsulinemia and hyperglycemia/hyperinsulinemia exhibited high levels of triglycerides (TGs) in the plasma and intestine and postprandial plasma chylomicrons and apo B-48 compared with normoglycemic/normoinsulinemic animals. In vitro studies, using cultured jejunal explants incubated with either [14C]oleic acid or [35S]methionine, revealed their higher efficiency in de novo TG synthesis, apo B-48 biogenesis, and TG-rich lipoprotein assembly. Accordingly, enhanced monoacylglycerol and diacylglycerol acyltransferase activity was also discernible and concomitant with an increased content of L-fatty acid binding protein and in vivo chylomicron production rates. However, both the I-fatty acid binding protein amount and the apo B-48 proteasomal degradative pathway were decreased. Overall, our findings show that the development of an insulin-resistant/diabetic state in Psammomys obesus triggers the whole intra-enterocyte machinery, leading to lipoprotein assembly and favoring the intestinal oversecretion of apo B-48-lipoproteins, which may contribute to characteristic hypertriglyceridemia.