-
[show abstract]
[hide abstract]
ABSTRACT: Leptin receptors (LEPR) are expressed in intestinal epithelial cells from the duodenum to the colon. Since their role is fundamental for the proper control of nutrient absorption, mucus secretion and mucosa renewal, the regulation of LEPR expression is for the first time investigated as a function of various potential effectors.
Fully differentiated Caco-2/15 cells were incubated for 24 hours with nutrients [carbohydrates, fatty acids (FA), amino acids and sterols], hormones (leptin, insulin, hydrocortisone and epithelial growth factor), inflammatory agents (Interferon-γ, LPS, TNF-α), and PPAR agonists (rosiglitazone and WY14643). Levels of LEPR mRNA and protein expressions were measured by RT-PCR and Western blots, respectively.
Long (219.1) and short (219.3) isoforms of the LEPR were detected in Caco-2/15 cells, while absence of the isoform 219.2 was noted. Their gene expression was modulated by carbohydrates, FA, PPAR agonists, biliary salts, insulin and leptin itself. On the other hand, LEPR protein expression was modulated by FA, cholesterol, biliary salts, PPAR agonists and insulin. Interestingly, the same effectors may have opposite effects on the short and the long LEPR isoforms, as well as on mRNA and protein levels. Finally, Caco-2/15 cells were found to be sensitive to the effector location, i.e. apical or basolateral compartment.
Our results suggest that (i) the expression of LEPR in Caco-2/15 cell line is not constitutive; (ii) the agents present in the apical or basolateral medium have different effects on LEPR mRNA and/or protein levels; and (iii) short and long isoforms of LEPR follow different patterns of regulation.
Endocrine, metabolic & immune disorders drug targets. 01/2012; 12(1):57-70.
-
[show abstract]
[hide abstract]
ABSTRACT: The Psammomys obesus lives in natural desert habitat on low energy (LE) diet, however when maintained in laboratory conditions with high energy (HE) diet it exhibits pathological metabolic changes resembling those of type 2 diabetes. We have evaluated and correlated the histopathology, metabolic and functional renal alterations occurring in the diabetic Psammomys. Renal function determined by measuring glomerular filtration rate (GFR), protein excretion, protein/creatinine ratio and morpho-immunocytochemical evaluations were performed on HE diet diabetic animals and compared to LE diet control animals. The diabetic animals present a 54% increase in GFR after one month of hyperglycemic condition and a decrease of 47% from baseline values after 4 months. Protein excretion in diabetic animals was 5 folds increased after 4 months. Light microscopy showed an increase in glomeruli size in the diabetic Psammomys, and electron microscopy and immunocytochemical quantitative evaluations revealed accumulation of basement membrane material as well as frequent splitting of the glomerular basement membrane. In addition, glycogen-filled Armanni-Ebstein clear cells were found in the distal tubules including the thick ascending limbs of the diabetic animals. These renal complications in the Psammomys, including changes in GFR with massive proteinuria sustained by physiological and histopathological changes, are very similar to the diabetic nephropathy in human. The Psamommys obesus represents therefore a reliable animal model of diabetic nephropathy.
Anatomy & cell biology 09/2011; 44(3):176-85.
-
[show abstract]
[hide abstract]
ABSTRACT: Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes.
Journal of Histochemistry and Cytochemistry 08/2011; 59(10):899-907. · 2.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The understanding of the regulation of food intake has become increasingly complex. More than 20 hormones, both orexigenic and anorexigenic, have been identified. After crossing the blood-brain barrier, they reach their main site of action located in several hypothalamic areas and interact to balance satiety and hunger. One of the most significant advances in this matter has been the discovery of leptin. This hormone plays fundamental roles in the control of appetite and in regulating energy expenditure. In accordance with the lipostatic theory stated by Kennedy in 1953, leptin was originally discovered in white adipose tissue. Its expression by other tissues was later established. Among them, the gastric mucosa has been shown to secrete large amounts of leptin. Both the adipose and the gastric tissues share similar characteristics in the synthesis and storage of leptin in granules, in the formation of a complex with the soluble receptor and a secretion modulated by hormones and energy substrates. However while adipose tissue secretes leptin in a slow constitutive endocrine way, the gastric mucosa releases leptin in a rapid regulated exocrine fashion into the gastric juice. Exocrine-secreted leptin survives the extreme hydrolytic conditions of the gastric juice and reach the duodenal lumen in an intact active form. Scrutiny into transport mechanisms revealed that a significant amount of the exocrine leptin crosses the intestinal wall by active transcytosis. Leptin receptors, expressed on the luminal and basal membrane of intestinal epithelial cells, are involved in the control of nutrient absorption by enterocytes, mucus secretion by goblet cells and motility, among other processes, and this control is indeed different depending upon luminal or basal stimulus. Gastric leptin after transcytosis reaches the central nervous system, to control food intake. Studies using the Caco-2, the human intestinal cell line, in vitro allowed analysis of the mechanisms of leptin actions on the intestinal mucosa, identification of the mechanisms of leptin transcytosis and understanding the modulation of leptin receptors by nutrients and hormones. Exocrine-secreted gastric leptin thus participates in a physiological axis independent in terms of time and regulation from that of adipose tissue to rapidly control food intake and nutrient absorption. Adipocytes and gastric epithelial cells are two cell types the metabolism of which is closely linked to food intake and energy storage. The coordinated secretion of adipose and gastric leptins ensures proper management of food processing and energy storage.
Progress in Histochemistry and Cytochemistry 09/2010; 45(3):143-200. · 4.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
International Journal of Cell Biology 01/2010; 2010:928169.
-
[show abstract]
[hide abstract]
ABSTRACT: Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK alpha and beta subunits were located both in the cytosol and in close association with rosettes of glycogen particles (alpha particles). To reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required. These included fixation with a combination of glutaraldehyde and paraformaldehyde, followed by postfixation with osmium tetroxide and lead citrate and embedding in Epon. Processing by less-stringent fixation conditions and embedding in Lowicryl led to the extraction of the glycogen deposits, which in turn resulted in the absence of any labeling. This indicates that the loss of glycogen deposits leads to the loss of closely associated proteins. Labeling for the alpha(1) and alpha(2) subunits of AMPK was found to be about 2-fold greater over glycogen than over cytosol, whereas labeling for beta(1) was 8-fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the beta(1) subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches.
Journal of Histochemistry and Cytochemistry 08/2009; 57(10):963-71. · 2.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Histochemie 07/2009; 132(3):351-67. · 2.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A major concern regarding the chronic administration of antiretroviral drugs is the potential for induction of drug efflux transporter expression (i.e., P-glycoprotein, P-gp) at tissue sites that can significantly affect drug distribution and treatment efficacy. Previous data have shown that the inductive effect of human immunodeficiency virus protease inhibitors (PIs) is mediated through the human orphan nuclear receptor, steroid xenobiotic receptor (SXR or hPXR). The objectives of this study were to investigate transport and inductive properties on efflux drug transporters of two PIs, atazanavir and ritonavir, at the blood-brain barrier by using a human brain microvessel endothelial cell line, hCMEC/D3. Transport properties of PIs by the drug efflux transporters P-gp and multidrug resistance protein 1 (MRP1) were assessed by measuring the cellular uptake of (3)H-atazanavir or (3)H-ritonavir in P-gp and MRP1 overexpressing cells as well as hCMEC/D3. Whereas the P-gp inhibitor, PSC833, increased atazanavir and ritonavir accumulation in hCMEC/D3 cells by 2-fold, the MRP inhibitor MK571 had no effect. P-gp, MRP1, and hPXR expression and localization were examined by Western blot analysis and immunogold cytochemistry at the electron microscope level. Treatment of hCMEC/D3 cells for 72 hr with rifampin or SR12813 (two well-established hPXR ligands) or PIs (atazanavir or ritonavir) resulted in an increase in P-gp expression by 1.8-, 6-, and 2-fold, respectively, with no effect observed for MRP1 expression. In hCMEC/D3 cells, cellular accumulation of these PIs appears to be primarily limited by P-gp efflux activity. Long-term exposure of atazanavir or ritonavir to brain microvessel endothelium may result in further limitations in brain drug permeability as a result of the up-regulation of P-gp expression and function.
Journal of Neuroscience Research 11/2008; 87(4):1023-36. · 2.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
AJP Gastrointestinal and Liver Physiology 09/2008; 295(5):G873-85. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Aging and diabetes are associated with exacerbated expression of adhesion molecules. Given their importance in endothelial dysfunction and their possible involvement in the alteration of glomerular permeability occurring in diabetes, we have evaluated expression of the sialomucin-type adhesion molecule CD34 in renal glomerular cells of normal and diabetic animals at two different ages by colloidal gold immunocytochemistry and immunoblotting. CD34 labeling was mostly assigned to the plasma membranes of glomerular endothelium and mesangial processes. Podocyte membranes were also labeled, but to a lesser degree. Short- and long-term diabetes triggers a substantial increase in immunogold labeling for CD34 in renal tissues compared with young normoglycemic animals. However, the level of labeling in old diabetic and healthy control rats is similar, suggesting that the effect of diabetes and aging on CD34 expression is similar but not synergistic. Western blotting of isolated glomerular fractions corroborated immunocytochemical results. Increased expression of CD34 may reflect its involvement in the pathogenesis of glomerular alterations related to age and diabetes. Alterations present in early diabetes, resembling those occurring with age, strengthen the concept that diabetes is an accelerated form of aging.
Journal of Histochemistry and Cytochemistry 07/2008; 56(6):605-14. · 2.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Novel approach in low voltage transmission electron microscopy (TEM) has revealed the presence of SV40 viral like particles in the secretory zymogen granules and in spherical membrane-bound dense bodies of SV40 infected pancreatic cells. The presence of SV40 antigen in these cellular compartments was confirmed by immunocytochemistry of the VP1 antigen. Visualization of the viral particles was only possible by examining ultrathin tissue sections with low-voltage TEM that significantly enhances imaging contrast. Results indicate that following infection of the cell entry and trafficking of the viral particles are present in unique cellular compartments such as ER, dense bodies, and secretory granules.
Microscopy Research and Technique 06/2008; 71(9):659-62. · 1.79 Impact Factor
-
Nilly Shimony, Moise Bendayan,
Gregory Elkin,
Orly Ben-nun-Shaul,
Mahmoud Abd-El-Latif,
Pnina Scherzer,
Ofer Arbel,
Ehud Ziv,
Lina Krasny,
Galina Pizov,
Ariella Oppenheim,
Yosef S Haviv
[show abstract]
[hide abstract]
ABSTRACT: Viral vector uptake into the pancreas is rare. The few viral vectors reported to transduce in vivo pancreatic islets after systemic injection required additional physical measures, such as direct pancreatic injection or hepatic vessel clamping. Because pancreatic islet uptake of the human polyomavirus family member BK virus was previously reported in hamsters after systemic administration, we hypothesized that SV40, a polyomavirus member remarkably similar to BK virus, may also infect the pancreas.
We injected intravenously a low dose of SV40, unaided by any other physical or chemical means, and evaluated viral uptake by pancreatic islets and pancreatic exocrine tissue via polymerase chain reaction, Western blot, electron microscopy, immunofluorescent microscopy, and protein A-gold immunocytochemistry.
Pancreatic uptake of SV40 was comparable to other major organs (ie, liver and spleen). SV40 viral particles were detected in both pancreatic islets and acini. In pancreatic islets, all islet cell types were infected by SV40, albeit the infection rate of glucagon-producing alpha cells surpassed beta- and delta-islet cells. Low-dose SV40 administration was not sufficient to induce heterologous gene expression in the pancreas.
Our study shows that pancreatic islet and acinar cell uptake of SV40 is feasible with a single, low-dose intravenous injection. However, this dose did not result in gene delivery into the murine pancreas.
Pancreas 06/2008; 36(4):411-6. · 2.39 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase 2 (PON2), a member of a powerful antioxidant family in human and rat intestine. Biochemical and ultrastructural experiments all showed a substantial expression of PON2 in human and rat intestine. Western blot analysis disclosed higher levels of PON2 in the jejunum than in the duodenum, ileum, and colon. Cell fractionation revealed a predominant PON2 association with microsomes and lysosomes in the human jejunum, which differed from that in rats. PON2 was detected in the intestine as early as week 15 of gestation and was significantly increased by week 20. Iron ascorbate-mediated lipid peroxidation induced a marked decrease in PON2 expression in intestinal specimens coincidental to an abundant rise in malondialdehyde (MDA). On the other hand, preincubation with potent antioxidants, such as butylated hydroxytoluene, Trolox, and N-acetylcysteine, prevented iron-ascorbate-generating PON2 reduction in parallel with MDA suppression. Finally, the preincubation of permeabilized Caco-2 cells with purified PON2 led to a protection against iron-ascorbate-induced lipid peroxidation. These observations demonstrate that the human intestine is preferentially endowed with a marked PON2 expression compared with the rat intestine and this expression shows a developmental and intracellular pattern of distribution. Furthermore, our observations suggest PON2 protective effects against prooxidant stimuli in the small intestine.
AJP Gastrointestinal and Liver Physiology 01/2008; 293(6):G1252-61. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: P-glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.
Biochemical and Biophysical Research Communications 10/2007; 361(2):301-6. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.
Journal of Histochemistry and Cytochemistry 11/2006; 54(10):1159-67. · 2.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 [(14)C]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.
The Journal of Lipid Research 11/2006; 47(10):2112-20. · 5.56 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum-Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.
Biochemistry and Cell Biology 05/2006; 84(2):207-14. · 2.67 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferation and viability. Studies using these transfected cells cultured with [14C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [35S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.
Biochemical and Biophysical Research Communications 02/2006; 339(1):248-54. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A role for cytosolic malonyl-CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl-CoA levels and of hepatic fatty acid oxidation.
FEBS Letters 01/2006; 579(29):6581-6. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Microsomal triglyceride transfer protein (MTP) is a heterodimeric complex consisting of a unique large 97-kDa protein and the multifunctional 58-kDa protein disulfide isomerase (PDI). It plays an essential role in the assembly of lipoproteins by shuttling lipids between phospholipid membranes. Based on cell fractionation, early studies have suggested the endoplasmic reticulum (ER) as the exclusive site of MTP. Focusing on the plasma membrane in this study, our attempts with immunoelectron microscopy and specific antibodies surprisingly revealed that labeling was not exclusively confined to the microsomes of rat absorptive cells. Immunogold labeling was also detected over the microvillus membrane of enterocytes. Western blot analysis and biochemical activity measurement confirmed MTP protein expression in brush-border membrane vesicles (BBMV) isolated from the intestinal epithelial cells of various species. Furthermore, MTP was coexpressed in microvilli membrane with PDI that is crucial to maintain the structure and activity of the MTP complex. The treatment of Caco-2 cells with nocodazole and colchicine blocked the appearance of MTP in the apical membrane. Similarly, the addition of BMS-197636, a known inhibitor of MTP transfer activity, suppressed the latter. In conclusion, the present studies suggest that MTP is present in the brush-border membrane of the enterocyte. Understanding the possible physiological role of MTP in this location may reveal additional functions.
Experimental Cell Research 11/2004; 300(1):11-22. · 3.58 Impact Factor
