Emilie Grenier

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.

Cholesterol content in brains of suicide completers

An association between low levels of serum cholesterol and violent or suicidal behaviour has frequently been reported, but it remains unclear how cholesterol in the peripheral system might be related to the brain functions involved in mediating suicidal behaviour. To our knowledge, there have been no previous studies aimed at answering the important question of whether there are differences in cholesterol within the brains of suicide completers. In the present study, cholesterol content was measured in cortical and subcortical tissue of brains from 41 male suicide completers and 21 male controls that died of sudden causes with no direct influence on brain tissue. No significant differences in cholesterol content were found between suicides and controls in the frontal cortex, amygdala or hippocampus. However, when the suicide completers were stratified into violent (n=31) or non-violent (n=10) groups based on the method of death, violent suicides were found to have lower grey-matter cholesterol content overall compared to non-violent suicides [F(1,111)=4.75, p=0.03], specifically in the frontal cortex (t=-4.16, d.f.=37, p<0.0005). Further exploration of the frontal cortex revealed that violent suicides had lower cholesterol content compared to non-violent suicides in the orbitofrontal cortex (t=-2.01, d.f.=36, p=0.05) and the ventral prefrontal cortex (t=-2.49, d.f.=37, p=0.02). This study represents the first direct examination of cholesterol content in brain tissue from suicide completers, and the present findings provide added support for the relationship between low cholesterol and violent or suicidal behaviour.

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.

Maternal and fetal leptin, adiponectin levels and associations with fetal insulin sensitivity

It remains uncertain whether leptin and adiponectin levels are correlated in maternal vs. fetal circulations. Little is known about whether leptin and adiponectin affect insulin sensitivity during fetal life.

Intestinal Lipid Handling Evidence and Implication of Insulin Signaling Abnormalities in Human Obese Subjects

Objective— Animal models have evidenced the role of intestinal triglyceride-rich lipoprotein overproduction in dyslipidemia. However, few studies have confronted this issue in humans and disclosed the intrinsic mechanisms. This work aimed to establish whether intestinal insulin resistance modifies lipid and lipoprotein homeostasis in the intestine of obese subjects. Approach and Results— Duodenal specimens obtained from 20 obese subjects undergoing bariatric surgery were paired for age, sex, and body mass index with or without insulin resistance, as defined by the homeostasis model assessment of insulin resistance. Insulin signaling, biomarkers of inflammation and oxidative stress, and lipoprotein assembly were assessed. The intestine of insulin-resistant subjects showed defects in insulin signaling as demonstrated by reduced protein kinase B phosphorylation and increased p38 mitogen-activated protein kinase phosphorylation, likely as the result of high oxidative stress (evidenced by malondialdehyde and conjugated dienes) and inflammation (highlighted by nuclear factor-κB, tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, and cyclooxygenase-2). Enhanced de novo lipogenesis rate and apolipoprotein B-48 biogenesis along with exaggerated triglyceride-rich lipoprotein production were observed, concomitantly with the high expression levels of liver and intestinal fatty acid–binding proteins and microsomal transfer protein. The presence of an aberrant intracellular cholesterol transport/metabolism was also suggested by the reduced expression of ATP-binding cassette A1 transporter and proprotein convertase subtilisin/kexin type 9. Conclusions— According to the present data, the small intestine may be classified as an insulin-sensitive tissue. Dysregulation of intestinal insulin signaling, possibly triggered by oxidative stress and inflammation, was associated with exaggerated lipogenesis and lipoprotein synthesis, which may represent a key mechanism for atherogenic dyslipidemia in patients with metabolic syndrome. # Significance {#article-title-60}Objective— Animal models have evidenced the role of intestinal triglyceride-rich lipoprotein overproduction in dyslipidemia. However, few studies have confronted this issue in humans and disclosed the intrinsic mechanisms. This work aimed to establish whether intestinal insulin resistance modifies lipid and lipoprotein homeostasis in the intestine of obese subjects. Approach and Results— Duodenal specimens obtained from 20 obese subjects undergoing bariatric surgery were paired for age, sex, and body mass index with or without insulin resistance, as defined by the homeostasis model assessment of insulin resistance. Insulin signaling, biomarkers of inflammation and oxidative stress, and lipoprotein assembly were assessed. The intestine of insulin-resistant subjects showed defects in insulin signaling as demonstrated by reduced protein kinase B phosphorylation and increased p38 mitogen-activated protein kinase phosphorylation, likely as the result of high oxidative stress (evidenced by malondialdehyde and conjugated dienes) and inflammation (highlighted by nuclear factor-&kgr;B, tumor necrosis factor-&agr;, interleukin-6, intercellular adhesion molecule-1, and cyclooxygenase-2). Enhanced de novo lipogenesis rate and apolipoprotein B-48 biogenesis along with exaggerated triglyceride-rich lipoprotein production were observed, concomitantly with the high expression levels of liver and intestinal fatty acid–binding proteins and microsomal transfer protein. The presence of an aberrant intracellular cholesterol transport/metabolism was also suggested by the reduced expression of ATP-binding cassette A1 transporter and proprotein convertase subtilisin/kexin type 9. Conclusions— According to the present data, the small intestine may be classified as an insulin-sensitive tissue. Dysregulation of intestinal insulin signaling, possibly triggered by oxidative stress and inflammation, was associated with exaggerated lipogenesis and lipoprotein synthesis, which may represent a key mechanism for atherogenic dyslipidemia in patients with metabolic syndrome.

Intestinal Lipid HandlingSignificance

Objective— Animal models have evidenced the role of intestinal triglyceride-rich lipoprotein overproduction in dyslipidemia. However, few studies have confronted this issue in humans and disclosed the intrinsic mechanisms. This work aimed to establish whether intestinal insulin resistance modifies lipid and lipoprotein homeostasis in the intestine of obese subjects. Approach and Results— Duodenal specimens obtained from 20 obese subjects undergoing bariatric surgery were paired for age, sex, and body mass index with or without insulin resistance, as defined by the homeostasis model assessment of insulin resistance. Insulin signaling, biomarkers of inflammation and oxidative stress, and lipoprotein assembly were assessed. The intestine of insulin-resistant subjects showed defects in insulin signaling as demonstrated by reduced protein kinase B phosphorylation and increased p38 mitogen-activated protein kinase phosphorylation, likely as the result of high oxidative stress (evidenced by malondialdehyde and conjugated dienes) and inflammation (highlighted by nuclear factor-κB, tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, and cyclooxygenase-2). Enhanced de novo lipogenesis rate and apolipoprotein B-48 biogenesis along with exaggerated triglyceride-rich lipoprotein production were observed, concomitantly with the high expression levels of liver and intestinal fatty acid–binding proteins and microsomal transfer protein. The presence of an aberrant intracellular cholesterol transport/metabolism was also suggested by the reduced expression of ATP-binding cassette A1 transporter and proprotein convertase subtilisin/kexin type 9. Conclusions— According to the present data, the small intestine may be classified as an insulin-sensitive tissue. Dysregulation of intestinal insulin signaling, possibly triggered by oxidative stress and inflammation, was associated with exaggerated lipogenesis and lipoprotein synthesis, which may represent a key mechanism for atherogenic dyslipidemia in patients with metabolic syndrome. # Significance {#article-title-60}Objective— Animal models have evidenced the role of intestinal triglyceride-rich lipoprotein overproduction in dyslipidemia. However, few studies have confronted this issue in humans and disclosed the intrinsic mechanisms. This work aimed to establish whether intestinal insulin resistance modifies lipid and lipoprotein homeostasis in the intestine of obese subjects. Approach and Results— Duodenal specimens obtained from 20 obese subjects undergoing bariatric surgery were paired for age, sex, and body mass index with or without insulin resistance, as defined by the homeostasis model assessment of insulin resistance. Insulin signaling, biomarkers of inflammation and oxidative stress, and lipoprotein assembly were assessed. The intestine of insulin-resistant subjects showed defects in insulin signaling as demonstrated by reduced protein kinase B phosphorylation and increased p38 mitogen-activated protein kinase phosphorylation, likely as the result of high oxidative stress (evidenced by malondialdehyde and conjugated dienes) and inflammation (highlighted by nuclear factor-&kgr;B, tumor necrosis factor-&agr;, interleukin-6, intercellular adhesion molecule-1, and cyclooxygenase-2). Enhanced de novo lipogenesis rate and apolipoprotein B-48 biogenesis along with exaggerated triglyceride-rich lipoprotein production were observed, concomitantly with the high expression levels of liver and intestinal fatty acid–binding proteins and microsomal transfer protein. The presence of an aberrant intracellular cholesterol transport/metabolism was also suggested by the reduced expression of ATP-binding cassette A1 transporter and proprotein convertase subtilisin/kexin type 9. Conclusions— According to the present data, the small intestine may be classified as an insulin-sensitive tissue. Dysregulation of intestinal insulin signaling, possibly triggered by oxidative stress and inflammation, was associated with exaggerated lipogenesis and lipoprotein synthesis, which may represent a key mechanism for atherogenic dyslipidemia in patients with metabolic syndrome.

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.

Oxidized low-density lipoproteins in cord blood from neonates with intra-uterine growth restriction

OBJECTIVE We verified whether oxidative stress indices (oxidized low-density lipoproteins and malondialdehyde) and inflammatory biomarkers (circulating C-reactive protein, interleukin-6, tumour necrosis factor-α, serum amyloid A and soluble intercellular vascular cell adhesion molecule) are increased in the umbilical vein of placental insufficiency induced intra-uterine growth restricted neonates. STUDY DESIGN The prospective cohort study, involving 3 tertiary care centers, consists of 200 consecutively recruited pregnant women carrying twins. We chose the twin pregnancy model because both fetuses share the same maternal environment, thereby avoiding potential confounding factors when comparing oxidative stress and inflammation biomarkers. We analysed only twin pairs with one with intra-uterine growth restriction (N=38) defined as fetal growth<10th percentile with abnormal Doppler of the umbilical artery. Blood samples were taken at birth from the umbilical vein. Intra-pair comparisons on the biomarkers were performed using the Student paired t-test. RESULTS We observed increased cord blood levels of oxidized low-density lipoproteins, (2.394 ± .412 vs 1.296 ± .204, p=.003) but not of malondialdehyde in growth restricted neonates when compared to their normal counterparts. Although indices of inflammation tended to be increased in cord blood from growth restricted newborns, the difference did not reach statistical significance. CONCLUSION In the twin model, intra-uterine growth restriction is associated with low-density lipoprotein oxidation without apparent dysregulation of inflammation biomarkers. CONDENSATION Increased oxidized low-density lipoproteins are observed in growth restricted twins compared to their co-twins with normal growth at birth.

Modulatory Role of PYY in Transport and Metabolism of Cholesterol in Intestinal Epithelial Cells

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 [14C]-acetate, [14C]-cholesterol, and [14C]-oleate, respectively. Biogenesis of apos (A-I, A-IV, E, B-48 and B-100) was examined by the incorporation of [35S]-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 [14C]-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.

Role of the apical and basolateral domains of the enterocyte in the regulation of cholesterol transport by a high glucose concentration

We have recently shown that a high glucose (HG) concentration raised intestinal cholesterol (CHOL) transport and metabolism in intestinal epithelial cells. The objective of the present work is to determine whether the stimulus for increased CHOL absorption by glucose originates from the apical site (corresponding to the intestinal lumen) or from the basolateral site (related to blood circulation). We tackled this issue by using differentiated Caco-2/15 cells. Only basolateral medium, supplemented with 25 mmol/L glucose, stimulated [(14)C]-CHOL uptake via the up-regulation of the critical CHOL transporter NPC1L1 protein, as confirmed by its specific ezetimibe inhibitor that abolished the rise in glucose-mediated CHOL capture. No significant changes were noted in SR-BI and CD36. Elevated CHOL uptake was associated with an increase in the transcription factors SREBP-2, LXR-β, and ChREBP, along with a fall in RXR-α. Interestingly, although the HG concentration in the apical medium caused modest changes in CHOL processing, its impact was synergetic with that of the basolateral medium. Our results suggest that HG concentration influences positively intestinal CHOL uptake when present in the basolateral medium. In addition, excessive consumption of diets containing high levels of carbohydrates may strengthen intestinal CHOL uptake in metabolic syndrome, thereby contributing to elevated levels of circulating CHOL and, consequently, the risk of developing type 2 diabetes and cardiovascular disease.