Geneviève Mailhot

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.

Localization, function and regulation of the two intestinal fatty acid-binding protein types.

Although intestinal (I) and liver (L) fatty acid binding proteins (FABP) have been widely studied, the physiological significance of the presence of the two FABP forms (I- and L-FABP) in absorptive cells remains unknown as do the differences related to their distribution along the crypt-villus axis, regional expression, ontogeny and regulation in the human intestine. Our morphological experiments supported the expression of I- and L-FABP as early as 13 weeks of gestation. Whereas cytoplasmic immunofluorescence staining of L-FABP was barely detectable in the lower half of the villus and in the crypt epithelial cells, I-FABP was visualized in epithelial cells of the crypt-villus axis in all intestinal segments until the adult period in which the staining was maximized in the upper part of the villus. Immunoelectron microscopy revealed more intense labeling of L-FABP compared with I-FABP, accompanied with a heterogeneous distribution in the cytoplasm, microvilli and basolateral membranes. By western blot analysis, I- and L-FABP at 15 weeks of gestation appeared predominant in jejunum compared with duodenum, ileum, proximal and distal colon. Exploration of the maturation aspect documented a rise in L-FABP in adult tissues. Permanent transfections of Caco-2 cells with I-FABP cDNA resulted in decreased lipid export, apolipoprotein (apo) biogenesis and chylomicron secretion. Additionally, supplementation of Caco-2 with insulin, hydrocortisone and epidermal growth factor differentially modulated the expression of I- and L-FABP, apo B-48 and microsomal triglyceride transfer protein (MTP), emphasizing that these key proteins do not exhibit a parallel modulation. Overall, our findings indicate that the two FABPs display differences in localization, regulation and developmental pattern.

Expression of and Role for Ovarian Cancer G-protein-coupled Receptor 1 (OGR1) during Osteoclastogenesis

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-κB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1tl/csf1tl) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.

Intestinal fatty acid binding protein regulates mitochondrion β-oxidation and cholesterol uptake

The role of intestinal fatty acid binding protein (I-FABP) in lipid metabolism remains elusive. To address this issue, normal human intestinal epithelial cells (HIEC-6) were transfected with cDNA to overexpress I-FABP and compared with cells treated with empty pQCXIP vector. I-FABP overexpression stimulated mitochondrial [U-14C]oleate oxidation to CO2 and acid-soluble metabolites via mechanisms including the upregulation of protein expression and the activity of carnitine palmitoyltransferase 1, a critical enzyme controlling the entry of fatty acid (FA) into mitochondria, and increased activity of 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial beta-oxidation enzyme. On the other hand, the gene and protein expression of the key enzymes FA synthase and acetyl-coenzyme A carboxylase 2 was decreased, suggesting diminished lipogenesis. Furthermore, I-FABP overexpression caused a decline in [14C]free cholesterol (CHOL) incorporation. Accordingly, a significant lessening was observed in the gene expression of Niemann Pick C1-Like 1, a mediator of CHOL uptake, along with an increase in the transcripts and protein content of ABCA1 and ABCG5/ABCG8, acting as CHOL efflux pumps. Furthermore, I-FABP overexpression resulted in increased levels of mRNA, protein mass, and activity of HMG-CoA reductase, the rate-limiting step in CHOL synthesis. Scrutiny of the nuclear receptors revealed augmented peroxisome proliferator-activated receptor alpha,gamma and reduced liver X receptor-alpha in HIEC-6 overexpressing I-FABP. Finally, I-FABP overexpression did not influence acyl-coenzyme A oxidase 1, which catalyzes the first rate-limiting step in peroxisomal FA beta-oxidation. Overall, our data suggest that I-FABP may influence mitochondrial FA oxidation and CHOL transport by regulating gene expression and interaction with nuclear receptors.

CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

Background Abnormal fatty acid composition (FA) in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. The mechanism(s) underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role. Methodology/Principal Findings The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7)/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL), isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARα, LXRα, LXRβ and RXRα). Conclusions/Significance Collectively, our results indicate that CFTR depletion may disrupt FA homeostasis in intestinal cells through alterations in FA uptake and transport combined with stimulation of lipogenesis that occurs by an LXR/RXR-independent mechanism. These findings exclude a contributing role of CFTR in CF-associated fat malabsorption.

BMP-5 Expression Increases During Chondrocyte Differentiation In Vivo and In Vitro and Promotes Proliferation and Cartilage Matrix Synthesis in Primary Chondrocyte Cultures

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short‐ear (se) mice, which have BMP‐5 mutations, a role for BMP‐5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP‐5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP‐5 or its inhibition by anti‐BMP‐5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP‐5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP‐5 expression increasing as cells differentiated, in contrast to other BMPs. BMP‐5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP‐5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho‐Smads. Anti‐BMP‐5 antibody inhibited the endogenous BMP‐5, reducing cell proliferation and phospho‐Smad nuclear accumulation. Together, the results demonstrate that BMP‐5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP‐5 loss‐of‐function mutations is discussed. J. Cell. Physiol. 214:56–64, 2008.

Oxidative stress and mitochondrial functions in the intestinal Caco-2/15 Cell Line

Background Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules. Methodology/Principal Findings The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 µM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities. Conclusions/Significance Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.

CFTR knockdown stimulates lipid synthesis and transport in intestinal Caco-2/15 cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel highly expressed in epithelial cells of the gastrointestinal tract. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease characterized by pancreatic insufficiency, fat malabsorption, and steatorrhea. Despite the administration of pancreatic enzymes to normalize malabsorption, CF patients still experienced lipid fecal loss, nutritional deficiencies, and abnormalities in serum lipid profile, suggesting the presence of intrinsic defects in the intestinal handling of nutrients. The objective of the present study was to assess the impact of CFTR gene knockdown on intracellular lipid metabolism of the intestinal Caco-2/15 cell line. Partial CFTR gene inactivation led to cellular lipid accretion of phospholipids, triglycerides, and cholesteryl esters. Likewise, secretion of these lipid fractions was significantly increased following CFTR gene manipulation. As expected from these findings, the output of triglyceride-rich lipoproteins showed the same increasing pattern. Investigation of the mechanisms underlying these changes revealed that CFTR knockdown resulted in raised levels of apolipoproteins in cells and media and microsomal transfer protein activity, two important factors for the efficient assembly and secretion of lipoproteins. Similarly, scrutiny of the enzymatic monoacylglycerol acyltransferase and diacylglycerol acyltransferase, which exhibit dynamic function in triacylglycerol resynthesis and chylomicron formation in enterocytes, revealed a significant augmentation in their activity. Conversely, cholesterol uptake mediated by Niemann-Pick C1 like 1, Scavenger Receptor Class B Type I, and ATP-binding cassette G8 remains unaffected by genetic modification of CFTR. Collectively, these results highlight the role played by CFTR in intestinal handling of lipids and may suggest that factors other than defective CFTR are responsible for the abnormal intracellular events leading to fat malabsorption in CF patients.

False-positive beta-galactosidase staining in osteoclasts by endogenous enzyme: studies in neonatal and month-old wild-type mice.

Escherichia coli β-galactosidase (β-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by β-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined β-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). β-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using β-gal as a marker gene.

Plasma zinc in adults with cystic fibrosis: correlations with clinical outcomes.

BACKGROUND Zinc status has been previously documented in cystic fibrosis (CF) infants, children and adolescents. However, despite the increasing life expectancy observed in CF populations, data regarding zinc status of CF adults are surprisingly lacking. The objectives of this study were to (1) characterize zinc status and (2) explore associations between zinc status and clinical outcomes of CF adult patients. METHODS A retrospective chart review was performed for patients who had their plasma zinc measured between 2009 and 2012. Data included demographics, clinical characteristics, biochemical parameters and co-morbid conditions. RESULTS A total of 304 CF patients were included in the study. These patients displayed a good nutritional status (mean BMI±SD: 22.7±3.5) and moderate lung disease (mean FEV1±SD: 66.3±22.2). Low plasma zinc concentration (<9.2μmol/L) was found in 68 out of 304 CF patients (22.4%). Compared to patients with normal zinc, those with low zinc had significantly lower forced vital capacity and forced expiratory volume in one second. 72% of CF adults with low zinc suffered from bone disease (vs 49% with normal zinc, p=0.037) and 79% had impaired glycemic status (vs 58%, p=0.016). Accordingly, negative correlations were found between plasma zinc and glucose (r=-0.139, p=0.0001), HbA1c (r=-0.237, p=0.0001) and fructosamine (r=-0.134, p=0.034). In multiple linear regression, albumin and glycemic status were significant predictors of plasma zinc. CONCLUSION Our data indicated that nearly one quarter of CF adults with good nutritional status and moderate lung disease had low plasma zinc concentration and that low zinc status was associated with worse clinical outcomes.