Alain Théophile Sané

Localization and role of NPC1L1 in cholesterol absorption in human intestine

Recent studies have documented the presence of Niemann-Pick C1-Like 1 (NPC1L1) in the small intestine and its capacity to transport cholesterol in mice and rats. The current investigation was undertaken to explore the localization and function of NPC1L1 in human enterocytes. Cell fractionation experiments revealed an NPC1L1 association with apical membrane of the enterocyte in human jejunum. Signal was also detected in lysosomes, endosomes, and mitochondria. Confirmation of cellular NPC1L1 distribution was obtained by immunocytochemistry. Knockdown of NPC1L1 caused a decline in the ability of Caco-2 cells to capture micellar [14C]free cholesterol. Furthermore, this NPC1L1 suppression resulted in increased and decreased mRNA levels and activity of HMG-CoA reductase, the rate-limiting step in cholesterol synthesis, and of ACAT, the key enzyme in cholesterol esterification, respectively. An increase was also noted in the transcriptional factor sterol-regulatory element binding protein that modulates cholesterol homeostasis. Efforts were devoted to define the impact of NPC1L1 knockdown on other mediators of cholesterol uptake. RT-PCR evidence is presented to show the significant decrease in the levels of scavenger receptor class B type I (SR-BI) with no changes in ABCA1, ABCG5, and cluster determinant 36 in NPC1L1-deficient Caco-2 cells. Together, our data suggest that NPC1L1 contributes to intestinal cholesterol homeostasis and possibly cooperates with SR-BI to mediate cholesterol absorption in humans.

PCSK9 plays a significant role in cholesterol homeostasis and lipid transport in intestinal epithelial cells

OBJECTIVES The proprotein convertase subtillisin/kexin type 9 (PCSK9) regulates cholesterol metabolism via degradation of low-density lipoprotein receptor (LDLr). Although PCSK9 is abundantly expressed in the intestine, limited data are available on its functions. The present study aims at determining whether PCSK9 plays important roles in cholesterol homeostasis and lipid transport in the gut. METHODS AND RESULTS Caco-2/15 cells were used allowing the exploration of the PCSK9 secretory route through the apical and basolateral compartments corresponding to intestinal lumen and serosal circulation, respectively. The output of PCSK9 occurred through the basolateral membrane, a site characterized by the location of LDLr. Co-immunoprecipitation studies indicated an association between PCSK9 and LDLr. Addition of purified recombinant wild type and D374Y gain-of function PCSK9 proteins to the basolateral medium was followed by a decrease in LDLr concomitantly with the accumulation of both forms of PCSK9. Furthermore, the latter caused a significant enhancement in cholesterol uptake also evidenced by a raised protein expression of cholesterol transporters NPC1L1 and CD36 without changes in SR-BI, ABCA1, and ABCG5/G8. Moreover, exogenous PCSK9 altered the activity of HMG-CoA reductase and acylcoenzyme A: cholesterol acyltransferase, and was able to enhance chylomicron secretion by positively modulating lipids and apolipoprotein B-48 biogenesis. Importantly, PCSK9 silencing led to opposite findings, which validate our data on the role of PCSK9 in lipid transport and metabolism. Moreover, PCSK9-mediated changes persisted despite LDLr knockdown. CONCLUSIONS These findings indicate that, in addition to its effect on LDLr, PCSK9 modulates cholesterol transport and metabolism, as well as production of apo B-containing lipoproteins in intestinal cells.

Modulation of intestinal cholesterol absorption by high glucose levels : impact on cholesterol transporters, regulatory enzymes, and transcription factors.

Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistant/Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [(14)C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose (P < 0.04). Because combination of 5 mM glucose with 20 mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 (P < 0.02) and concomitantly decreased SR-BI protein mass (P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased (P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-alpha, LXR-beta, PPAR-beta, and PPAR-gamma along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes.

Increased hepatic lipogenesis in insulin resistance and Type 2 diabetes is associated with AMPK signalling pathway up-regulation in Psammomys obesus

AMPK (AMP-activated protein kinase) has been suggested to be a central player regulating FA (fatty acid) metabolism through its ability to regulate ACC (acetyl-CoA carboxylase) activity. Nevertheless, its involvement in insulin resistance- and TD2 (Type 2 diabetes)-associated dyslipidaemia remains enigmatic. In the present study, we employed the Psammomys obesus gerbil, a well-established model of insulin resistance and TD2, in order to appreciate the contribution of the AMPK/ACC pathway to the abnormal hepatic lipid synthesis and increased lipid accumulation in the liver. Our investigation provided evidence that the development of insulin resistance/diabetic state in P. obesus is accompanied by (i) body weight gain and hyperlipidaemia; (ii) elevations of hepatic ACC-Ser79 phosphorylation and ACC protein levels; (iii) a rise in the gene expression of cytosolic ACC1 concomitant with invariable mitochondrial ACC2; (iv) an increase in hepatic AMPKalpha-Thr172 phosphorylation and protein expression without any modification in the calculated ratio of phospho-AMPKalpha to total AMPKalpha; (v) a stimulation in ACC activity despite increased AMPKalpha phosphorylation and protein expression; and (vi) a trend of increase in mRNA levels of key lipogenic enzymes [SCD-1 (stearoyl-CoA desaturase-1), mGPAT (mitochondrial isoform of glycerol-3-phosphate acyltransferase) and FAS (FA synthase)] and transcription factors [SREBP-1 (sterol-regulatory-element-binding protein-1) and ChREBP (carbohydrate responsive element-binding protein)]. Altogether, our findings suggest that up-regulation of the AMPK pathway seems to be a natural response in order to reduce lipid metabolism abnormalities, thus supporting the role of AMPK as a promising target for the treatment of TD2-associated dyslipidaemia.

Expression of Sar1b Enhances Chylomicron Assembly and Key Components of the Coat Protein Complex II System Driving Vesicle Budding

Objective—SAR1b plays a significant role in the assembly, organization, and function of the coat protein complex II, a critical complex for the transport of proteins from the endoplasmic reticulum to the Golgi. Recently, mutations in SARA2 have been associated with lipid absorption disorders. However, functional studies on Sar1b-mediated lipid synthesis pathways and lipoprotein packaging have not been performed. Methods and Results—Sar1b was overexpressed in Caco-2/15 cells and resulted in significantly augmented triacylglycerol, cholesteryl ester, and phospholipid esterification and secretion and markedly enhanced chylomicron production. It also stimulated monoacylglycerol acyltransferase/diacylglycerol acyltransferase activity and enhanced apolipoprotein B-48 protein synthesis, as well as elevated microsomal triglyceride transfer protein activity. Along with the enhanced chylomicrons, microsomes were characterized by abundant Sec12, the guanine exchange factor that promotes the localization of Sar1b in the endoplasmic reticulum. Furthermore, coimmunoprecipitation experiments revealed high levels of the complex components Sec23/Sec24 and p125, the Sec23-interacting protein. Finally, a pronounced interaction of Sec23/Sec24 with sterol regulatory element binding protein (SREBP) cleavage-activating protein and SREBP-1c was noted, thereby permitting the transfer of the transcription factor SREBP-1c to the nucleus for the activation of genes involved in lipid metabolism. Conclusion—Our data suggest that Sar1b expression may promote intestinal lipid transport with the involvement of the coat protein complex II network and the processing of SREBP-1c.

AMPK in the Small Intestine in Normal and Pathophysiological Conditions

The role of AMPK in regulating energy storage and depletion remains unexplored in the intestine. This study will to define its status, composition, regulation and lipid function, as well as to examine the impact of insulin resistance and type 2 diabetes on intestinal AMPK activation, insulin sensitivity, and lipid metabolism. Caco-2/15 cells and Psammomys obesus (P. obesus) animal models were experimented. We showed the predominance of AMPKα1 and the prevalence of α1/β2/γ1 heterotrimer in Caco-2/15 cells. The activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside and metformin resulted in increased phospho(p)-ACC. However, the down-regulation of p-AMPK by compound C and high glucose lowered p-ACC without affecting 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Administration of metformin to P. obesus with insulin resistance and type 2 diabetes led to 1) an up-regulation of intestinal AMPK signaling pathway typified by ascending p-AMPKα(-Thr172); 2) a reduction in ACC activity; 3) an elevation of carnitine palmitoyltransferase 1; 4) a trend of increase in insulin sensitivity portrayed by augmentation of p-Akt and phospho-glycogen synthetase kinase 3β; 5) a reduced phosphorylation of p38-MAPK and ERK1/2; and 6) a decrease in diabetic dyslipidemia following lowering of intracellular events that govern lipoprotein assembly. These data suggest that AMPK fulfills key functions in metabolic processes in the small intestine.

Receptor-Mediated Transcytosis of Leptin through Human Intestinal Cells In Vitro

Gastric Leptin is absorbed by duodenal enterocytes and released on the basolateral side towards the bloodstream. We investigated in vitro some of the mechanisms of this transport. Caco-2/15 cells internalize leptin from the apical medium and release it through transcytosis in the basal medium in a time- temperature-dependent and saturable fashion. Leptin receptors are revealed on the apical brush-border membrane of the Caco-2 cells. RNA-mediated silencing of the receptor led to decreases in the uptake and basolateral release. Leptin in the basal medium was found bound to the soluble form of its receptor. An inhibitor of clathrin-dependent endocytosis (chlorpromazine) decreased leptin uptake. Confocal immunocytochemistry and the use of brefeldin A and okadaic acid revealed the passage of leptin through the Golgi apparatus. We propose that leptin transcytosis by intestinal cells depends on its receptor, on clathrin-coated vesicles and transits through the Golgi apparatus.

Sar1b transgenic male mice are more susceptible to high-fat diet-induced obesity, insulin insensitivity and intestinal chylomicron overproduction ☆

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.

Tissue distribution and regulation of the small Sar1b GTPase in mice.

Background/Aims: Sar1b GTPase (Sar1b) represents an obligatory component of COPII vesicles that bud from the endoplasmic reticulum to transport proteins to the Golgi apparatus. Its genetic mutations lead to a severe disorder known as chylomicron retention disease. Despite growing knowledge on Sar1b, little is known about it tissue distribution and regulation, which constitute the aims of the present study. We aimed to determine the Sar1b tissue distribution and modulation by a high-fat diet and gene forcing using transgenic mice in comparison to wild-type mice. Methods: The expression pattern of Sar1b was studied in different organs of wild-type and Sar1b transgenic mice by qRT-PCR and Western blot. The effect of transgenesis and insulin resistance induced by a 12-week high-fat diet on Sar1b gene expression was also assessed by qRT-PCR. Results: Evaluation of Sar1b mRNA revealed the skeletal muscle as the tissue with the highest Sar1b expression, followed by the heart and liver, the organs composing the digestive tract, the brain and finally the lung and the adipose tissue. Sar1b protein expression levels follow a similar pattern among the organs, except for its lower expression in the heart. While the high-fat diet did not exert any significant alterations, Sar1b transgenic mice displayed higher gene expression in the liver, ileum, jejunum, proximal and distal colon compared to wild-type mice. Conclusion: Our study supports the importance of Sar1b in organs involved in lipid transport and/or calcium trafficking such as the liver, intestine, skeletal muscle and heart.

Targeted CFTR gene disruption with zinc-finger nucleases in human intestinal epithelial cells induces oxidative stress and inflammation.

UNLABELLED Cystic fibrosis (CF) is a multisystemic pathology caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. OBJECTIVES As the intestine harbors the greatest number of CFTR transcripts after birth and since CFTR plays a role in glutathione transport, we hypothesized that CFTR deletion might produce oxidative stress (OxS) and inflammation in CF intestinal epithelial cell. METHODS CFTR gene was abrogated in Caco-2/15 enterocytes through the zinc-finger nuclease system. Their oxidative and inflammatory characteristics were appreciated under basal conditions and after the treatment with the pro-oxidant iron-ascorbate (Fe/Asc) complex and pro-inflammatory lipopolysaccharide (LPS). RESULTS Intestinal epithelial cells with CFTR knockout spontaneously exhibited an increased lipid peroxidation level, reflected by malondialdehyde overproduction and reduced antioxidant defense characterized by low enzymatic activities of glutathione peroxidase and catalase. CFTR silencing also resulted in elevated protein expression of pro-inflammatory tumor necrosis Factor-α, interleukin-6, cyclooxygenase-2, and the transcription factor nuclear factor-κB. Moreover, exaggerated OxS and inflammation processes occurred in CFTR(-/-) cells in response to the addition of Fe/Asc and LPS, respectively. CONCLUSIONS Intestinal Caco-2/15 cells with CFTR deletion, display innate oxidative and inflammatory features while being more sensitive to pro-oxidant and pro-inflammatory stimuli. These two pathophysiological processes could be implicated in CF-related intestinal disorders.