The small intestine is the essential site for the transport of alimentary fat in the form of lipoproteins. After the digestive phase, the lipolytic products in the lumen are absorbed by enterocytes that possess the unique ability to elaborate chylomicrons (CMs), the largest triglyceride (TG)-rich lipoprotein and dietary lipid vehicle (1, 2). CM assembly within epithelial absorptive cells is a multistep pathway that includes the translocation of lipolytic products from the apical membrane to the ER by cytosolic fatty acidbinding proteins (FABPs), lipid esterification, synthesis, and the posttranslational modification of different apos, especially apoB-48, as well as the packaging of lipid and apo components into pre-CM particles (3-5). It is only under these specific conditions that CM particles move huge amounts of dietary fat into the blood circulation via the lymphatic system. Accidents of nature reveal the intracellular roles of key proteins in CM assembly and secretion (6). For example, deciphering chylomicron retention disease (CRD) afforded new insight into the crucial functions of SAR1B GTPase, the defects of which lead to severe fat malabsorption, hypocholesterolemia, extensive steatorrhea, and significant failure to thrive in children (7, 8) with developmental abnormalities in various organs (9). It is now well established that mutations in SAR1B (SARA2) gene encoding the SAR1B GTPase protein prevent the coat protein complex II (COPII) from producing mature CM-contained vesicles endowed with the ability to bud from the ER and reach the Abstract Genetic defects in SAR1B GTPase inhibit chylomicron (CM) trafficking to the Golgi and result in a huge intraenterocyte lipid accumulation with a failure to release CMs and liposoluble vitamins into the blood circulation. The central aim of this study is to test the hypothesis that SAR1B deletion (SAR1B /) disturbs enterocyte lipid homeostasis (e.g., FA -oxidation and lipogenesis) while promoting oxidative stress and inflammation. Another issue is to compare the impact of SAR1B / to that of its paralogue SAR1A / and combined SAR1A / /B /. To address these critical issues, we have generated Caco-2/15 cells with a knockout of SAR1A, SAR1B, or SAR1A/B genes. SAR1B / results in lipid homeostasis disruption, reflected by enhanced mitochondrial FA -oxidation and diminished lipogenesis in intestinal absorptive cells via the implication of PPAR and PGC1 transcription factors. Additionally, SAR1B / cells, which mimicked enterocytes of CM retention disease, spontaneously disclosed inflammatory and oxidative characteristics via the implication of NF-B and NRF2. In most conditions, SAR1A / cells showed a similar trend, albeit less dramatic, but synergetic effects were observed with the combined defects of the two SAR1 paralogues. In conclusion, SAR1B and its paralogue are needed not only for CM trafficking but also for lipid homeostasis, prooxidant/antioxidant balance, and protection against inflammatory processes.-Sane, A., L. Ahmarani, E. Delvin, N. Aucl...

Chylomicron retention disease (CRD) is an autosomal recessive disorder associated with biallelic Sar1b mutations leading to defects in intracellular chylomicron (CM) trafficking and secretion. To date, a direct cause-effect relationship between CRD and Sar1b mutation has not been established, but genetically modified animal models provide an opportunity to elucidate unrecognized aspects of these mutations. To examine the physiological role and molecular mechanisms of Sar1b function, we generated mice expressing either a targeted deletion or mutation of human Sar1b using the CRISPR-Cas9 system. We found that deletion or mutation of Sar1b in mice resulted in late-gestation lethality of homozygous embryos. Moreover, compared with WT mice, heterozygotes carrying a single disrupted Sar1b allele displayed lower plasma levels of triglycerides, total cholesterol, and HDL-cholesterol, along with reduced CM secretion following gastric lipid gavage. Similarly, decreased expression of apolipoprotein B and microsomal triglyceride transfer protein was observed in correlation with the accumulation of mucosal lipids. Inefficient fat absorption in heterozygotes was confirmed via an increase in fecal lipid excretion. Furthermore, genetically modified Sar1b affected intestinal lipid homeostasis as demonstrated by enhanced fatty acid β-oxidation and diminished lipogenesis through the modulation of transcription factors. This is the first reported mammalian animal model with human Sar1b genetic defects, which reproduces some of the characteristic CRD features and provides a direct cause-effect demonstration.Supplementary key words Sar1b • gene defects • embryonic lethality • chylomicron • dietary fat • lipid metabolism • CRISPR/ Cas9 • chylomicron retention disease • fatty acid β-oxidation • lipogenesis

Our previous work showed that ET-1 induced a concentration-dependent increase of cytosolic Ca2+ ([Ca]c) and nuclear Ca2+ ([Ca]n) in human aortic vascular smooth muscle cells (hVSMCs). In the present study, using hVSMCs and 3-dimensional confocal microscopy coupled to the Ca2+ fluorescent probe Fluo-3, we showed that peptidic antagonists of ETA and ETB receptors (BQ-123 (10(-6) mol/L) and BQ-788 (10(-7) mol/L), respectively) prevented, but did not reverse, ET-1-induced sustained increase of [Ca]c and [Ca]n. In contrast, nonpeptidic antagonists of ETA and ETB (respectively, BMS-182874 (10(-8)-10(-6) mol/L) and A-192621 (10(-7) mol/L)) both prevented and reversed ET-1-induced sustained increase of [Ca]c and [Ca]n. Furthermore, activation of the ETB receptor alone using the specific agonist IRL-1620 (10(-9) mol/L) induced sustained increases of [Ca]c and [Ca]n, and subsequent administration of ET-1 (10(-7) mol/L) further increased nuclear Ca2+. ET-1-induced increase of [Ca]c and [Ca]n was completely blocked by extracellular application of the Ca2+ chelator EGTA. Pretreatment with the G protein inhibitors pertussis toxin (PTX) and cholera toxin (CTX) also prevented the ET-1 response; however, strong membrane depolarization with KCl (30 mmol/L) subsequently induced sustained increase of [Ca]c and [Ca]n. Pretreatment of hVSMCs with either the PKC activator phorbol-12,13-dibutyrate or the PKC inhibitor bisindolylmaleimide did not affect ET-1-induced sustained increase of intracellular Ca2+. These results suggest that both ETA- and ETB-receptor activation contribute to ET-1-induced sustained increase of [Ca]c and [Ca]n in hVSMCs. Moreover, in contrast to the peptidic antagonists of ET-1 receptors, the nonpeptidic ETA-receptor antagonist BMS-182874 and the nonpeptidic ETB-receptor antagonist A-192621 were able to reverse the effect of ET-1. Nonpeptidic ETA- and ETB-receptor antagonists may therefore be better pharmacological tools for blocking ET-1-induced sustained increase of intracellular Ca2+ in hVSMCs. Our results also suggest that the ET-1-induced sustained increase of [Ca]c and [Ca]n is not mediated via activation of PKC, but via a PTX- and CTX-sensitive G protein calcium influx through the R-type Ca2+ channel.