Schohraya Spahis

Intestinal cholesterol transport proteins: an update and beyond.

Purpose of review Various studies have delineated the causal role of dietary cholesterol in atherogenesis. Strategies have thus been developed to minimize cholesterol absorption, and cholesterol transport proteins found at the apical membrane of enterocytes have been extensively investigated. This review focuses on recent progress related to various brush-border proteins that are potentially involved in alimentary cholesterol transport. Recent findings Molecular mechanisms responsible for dietary cholesterol and plant sterol uptake have not been completely defined. Growing evidence, however, supports the concept that several proteins are involved in mediating intestinal cholesterol transport, including SR-BI, NPC1L1, CD36, aminopeptidase N, P-glycoprotein, and the caveolin-1/annexin-2 heterocomplex. Other ABC family members (ABCA1 and ABCG5/ABCG8) act as efflux pumps favoring cholesterol export out of absorptive cells into the lumen or basolateral compartment. Several of these cholesterol carriers influence intracellular cholesterol homeostasis and are controlled by transcription factors, including RXR, LXR, SREBP-2 and PPARα. The lack of responsiveness of NPC1L1-deficient mice to ezetimibe suggests that NPC1L1 is likely to be the principal target of this cholesterol-lowering drug. Summary The understanding of the role, genetic regulation and coordinated function of proteins mediating intestinal cholesterol transport may lead to novel ways of treating cardiovascular disease.

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.

Lipid profile, fatty acid composition and pro- and anti-oxidant status in pediatric patients with attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is the most prevalent behavioral disorder in children and the pathophysiology remains obscure. In addition to the pharmacotherapy, which is the primary treatment of ADHD, nutritional intervention may have a significant impact on ADHD symptoms. We studied lipid and lipoprotein profiles, fatty acid (FA) composition, and oxidant-antioxidant status in 37 pediatric ADHD patients and 35 healthy control subjects. Our results show that plasma triacylglycerols and phospholipids were lower, whereas free cholesterol, HDL, and apolipoprotein A-I were higher in ADHD patients compared with controls. The proportion of plasma EPA and DHA was higher, but that of oleic and alpha-linolenic (ALA) acids was lower. As expected from these findings, the proportions of both total saturates and polyunsaturates fatty acids (PUFA) were higher and lower, respectively, in ADHD patients than in controls, which led to a significant decrease in the PUFAs/saturates ratio. On the other hand, the ratios of eicosatrienoic acid to arachidonic acid and of palmitoleic acid to linoleic acid, established indexes of essential fatty acid (EFA) status remained unchanged revealing that EFA did not affect ADHD patients. Similarly, the activity of delta-6 desaturase, estimated by the ratio of 18:2(n-6)/20:4(n-6), was found unaffected, whereas ALA/EPA was diminished. Lessened lipid peroxidation was noted in ADHD subjects as documented by the diminished values of plasma malondialdehyde accompanied by increased concentrations of gamma-tocopherol. In conclusions, significant changes occur in the lipid and lipoprotein profiles, as well as in the oxidant-antioxidant status of ADHD patients, however, the FA distribution does not reflect n-3 FA deficiency.

Oxidative Stress as a Critical Factor in Nonalcoholic Fatty Liver Disease Pathogenesis

SIGNIFICANCE Nonalcoholic fatty liver disease (NAFLD), characterized by liver triacylglycerol build-up, has been growing in the global world in concert with the raised prevalence of cardiometabolic disorders, including obesity, diabetes, and hyperlipemia. Redox imbalance has been suggested to be highly relevant to NAFLD pathogenesis. Recent Advances: As a major health problem, NAFLD progresses to the more severe nonalcoholic steatohepatitis (NASH) condition and predisposes susceptible individuals to liver and cardiovascular disease. Although NAFLD represents the predominant cause of chronic liver disorders, the mechanisms of its development and progression remain incompletely understood, even if various scientific groups ascribed them to the occurrence of insulin resistance, dyslipidemia, inflammation, and apoptosis. Nevertheless, oxidative stress (OxS) more and more appears as the most important pathological event during NAFLD development and the hallmark between simple steatosis and NASH manifestation. CRITICAL ISSUES The purpose of this article is to summarize recent developments in the understanding of NAFLD, essentially focusing on OxS as a major pathogenetic mechanism. Various attempts to translate reactive oxygen species (ROS) scavenging by antioxidants into experimental and clinical studies have yielded mostly encouraging results. FUTURE DIRECTIONS Although augmented concentrations of ROS and faulty antioxidant defense have been associated to NAFLD and related complications, mechanisms of action and proofs of principle should be highlighted to support the causative role of OxS and to translate its concept into the clinic. Antioxid. Redox Signal. 26, 519-541.

Circulating docosahexaenoic acid levels are associated with fetal insulin sensitivity.

Background Arachidonic acid (AA; C20∶4 n-6) and docosahexaenoic acid (DHA; C22∶6 n-3) are important long-chain polyunsaturated fatty acids (LC-PUFA) in maintaining pancreatic beta-cell structure and function. Newborns of gestational diabetic mothers are more susceptible to the development of type 2 diabetes in adulthood. It is not known whether low circulating AA or DHA is involved in perinatally “programming” this susceptibility. This study aimed to assess whether circulating concentrations of AA, DHA and other fatty acids are associated with fetal insulin sensitivity or beta-cell function, and whether low circulating concentrations of AA or DHA are involved in compromised fetal insulin sensitivity in gestational diabetic pregnancies. Methods and Principal Findings In a prospective singleton pregnancy cohort, maternal (32-35 weeks gestation) and cord plasma fatty acids were assessed in relation to surrogate indicators of fetal insulin sensitivity (cord plasma glucose-to-insulin ratio, proinsulin concentration) and beta-cell function (proinsulin-to-insulin ratio) in 108 mother-newborn pairs. Cord plasma DHA levels (in percentage of total fatty acids) were lower comparing newborns of gestational diabetic (n = 24) vs. non-diabetic pregnancies (2.9% vs. 3.5%, P = 0.01). Adjusting for gestational age at blood sampling, lower cord plasma DHA levels were associated with lower fetal insulin sensitivity (lower glucose-to-insulin ratio, r = 0.20, P = 0.036; higher proinsulin concentration, r = −0.37, P <0.0001). The associations remained after adjustment for maternal and newborn characteristics. Cord plasma saturated fatty acids C18∶0 and C20∶0 were negatively correlated with fetal insulin sensitivity, but their levels were not different between gestational diabetic and non-diabetic pregnancies. Cord plasma AA levels were not correlated with fetal insulin sensitivity. Conclusion Low circulating DHA levels are associated with compromised fetal insulin sensitivity, and may be involved in perinatally “programming” the susceptibility to type 2 diabetes in the offspring of gestational diabetic mothers.

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.

Intestinal and Hepatic Cholesterol Carriers in Diabetic Psammomys obesus

Insulin resistance and type 2 diabetes (T2D) are characterized by hyperlipidemia. The aim of the present study was to elucidate whether T2D contributes to abnormal cholesterol (CHOL) homeostasis. Experiments were carried out in the small intestine and liver of Psammomys obesus, a model of nutritionally induced T2D. Our results show that diabetic animals exhibited a lower intestinal CHOL uptake, which was associated with a decrease in 1) the gene and protein expression of Niemann-Pick C1 like 1 that plays a pivotal role in CHOL incorporation in the enterocytes; and 2) mRNA of ATP-binding cassette transporters (ABC)A1 that mediates CHOL efflux from intestinal cells to apolipoprotein A-I and high-density lipoprotein. No changes were observed in the other intestinal transporters scavenger receptor-class B type I (SR-BI) and annexin 2. On the other hand, in diabetic animals, a significant mRNA decrease was noticed in intestinal ABCG5 and ABCG8 responsible for the secretion of absorbed CHOL back into the lumen. Furthermore, jejunal PCSK9 protein was diminished and low-density lipoprotein receptor was raised, along with a significant down-regulation in jejunal 3-hydroxy-3-methylglutaryl-coenzyme A reductase in P. obesus with T2D. Finally, among the transcription factors tested, only an increase in liver X receptors alpha and a decrease in peroxisome proliferator-activated receptors delta/beta mRNAs were detected in the intestine. In the liver, there was 1) an augmentation in the protein mass of Niemann-Pick C1 like 1, SR-BI, and annexin 2; 2) an up-regulation of SR-BI mRNA; 3) a fall in ABCG8 protein content as well as in ABCG5 and ABCA1 mRNA; and 4) an augmentation in liver X receptors alpha and peroxisome proliferator-activated receptors beta/delta mRNA, together with a drop in sterol regulatory element binding protein-2 protein. Our findings show that the development in P. obesus with T2D modifies the whole intraenterocyte and hepatocyte machinery responsible for CHOL homeostasis.

Plasma fatty acid composition in French-Canadian children with non-alcoholic fatty liver disease: Effect of n-3 PUFA supplementation

Non-alcoholic fatty liver disease (NAFLD) represents one of the most common causes of liver disease worldwide. As the NAFLD pathogenesis is associated with diet and lifestyle, the aims of the present work are to assess fatty acid (FA) composition in NAFLD young French-Canadian, to determine whether treatment with n-3 FA improves the plasma FA profile, and to define the time on the effectiveness of n-3 FA supplementation. Baseline characteristics of the NAFLD subjects show increased, anthropometric and biochemical parameters. Their plasma FA composition is characterized by a percent increase in total n-6 FA and a high proportion of saturated and total monounsaturated FA, as well as a decrease in Δ5 and increase in Δ6 desaturases. In conclusion, our results document for the first time the composition of plasma FAs in NAFLD young French Canadian and the efficacy of 3-month supplementation to improve the proportion of n-3 FA in their plasma.

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.

CFTR silencing in pancreatic β-cells reveals a functional impact on glucose-stimulated insulin secretion and oxidative stress response

Cystic fibrosis (CF)-related diabetes (CFRD) has become a critical complication that seriously affects the clinical outcomes of CF patients. Although CFRD has emerged as the most common nonpulmonary complication of CF, little is known about its etiopathogenesis. Additionally, whether oxidative stress (OxS), a common feature of CF and diabetes, influences CFRD pathophysiology requires clarification. The main objective of this study was to shed light on the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in combination with OxS in insulin secretion from pancreatic β-cells. CFTR silencing was accomplished in MIN6 cells by stable expression of small hairpin RNAs (shRNA), and glucose-induced insulin secretion was evaluated in the presence and absence of the valuable prooxidant system iron/ascorbate (Fe/Asc; 0.075/0.75 mM) along with or without the antioxidant Trolox (1 mM). Insulin output from CFTR-silenced MIN6 cells was significantly reduced (∼ 70%) at basal and at different glucose concentrations compared with control Mock cells. Furthermore, CFTR silencing rendered MIN6 cells more sensitive to OxS as evidenced by both increased lipid peroxides and weakened antioxidant defense, especially following incubation with Fe/Asc. The decreased insulin secretion in CFTR-silenced MIN6 cells was associated with high levels of NF-κB (the major participant in inflammatory responses), raised apoptosis, and diminished ATP production in response to the Fe/Asc challenge. However, these defects were alleviated by the addition of Trolox, thereby pointing out the role of OxS in aggravating the effects of CFTR deficiency. Our findings indicate that CFTR deficiency in combination with OxS may contribute to endocrine cell dysfunction and insulin secretion, which at least in part may explain the development of CFRD.