Fatty Acid Percentage Distribution in Complex Lipids of Breast Milk From Mothers on a Low Docosahexaenoic Acid Diet.

The aim of this study was to assess the fatty acid (FA) percentage distribution in complex lipids of breast milk from mothers on a low docosahexaenoic acid (DHA) diet. We performed a descriptive, cross-sectional study of milk samples (n = 14) collected 90 days after delivery and analyzed them using gas chromatography, thin-layer chromatography, and the Fiske-Subbarow method. Complex lipid distribution was 40.70 ± 5.11% sphingomyelin (SM), 26.03 ± 5.98% phosphatidylethanolamine (PE), 21.12 ± 2.32% phosphatidylcholine, 7.94 ± 1.96% phosphatidylserine, and 4.22 ± 1.25% phosphatidylinositol. Median DHA and arachidonic acid values were 0.13% (0.12; 0.18) and 0.42% (0.33; 0.53), respectively. Mean FA percentage in SM and PE was as follows: palmitic acid, 34.45 ± 1.94% and 5.38 ± 0.94%; oleic acid, 16.50 ± 4.07% and 9.43 ± 4.05%; linoleic acid, 5.91 ± 4.69% and 9.05 ± 4.5%. DHA was not detectable in SM, but it was found in PE (55.33 ± 14.46). In conclusion, breast milk of mothers on a low DHA diet contained 55% DHA in PE, but no DHA in SM.

Sphingosine-1-phosphate receptor 2 plays a dual role depending on the stage of cell differentiation in renal epithelial cells.

Epithelial renal cells have the ability to adopt different cellular phenotypes through epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). These processes are increasingly recognized as important repair factors following acute renal tubular injury. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with impact on proliferation, growth, migration, and differentiation which has significant implication in various diseases including cancer and kidney fibrosis. Here we demonstrated that S1P can exert by activating S1P receptor 2 (S1PR2) different functions depending on the stage of cell differentiation. We observed that the differences in the migratory profile of Madin-Darby canine kidney (MDCK) cells depend both on their stage of cell differentiation and the activity of S1PR2, a receptor that can either promote or inhibit the migratory process. Meanwhile in non-differentiated cells S1PR2 activation avoids migration, it is essential on fully differentiated cells. This is the first time that an antagonist effect of S1PR2 was reported for the same cell type. Moreover, in fully differentiated cells, S1PR2 activation is crucial for the progression of EMT - characterized by adherent junctions disassembly, ß-catenin and SNAI2 nuclear translocation and vimentin expression- and depends on ERK 1/2 activation and nuclear translocation. These findings provide a new perspective about the different S1PR2 functions depending on the stage of cell differentiation that can be critical to the modulation of renal epithelial cell plasticity, potentially paving the way for innovative research with pathophysiologic relevance.

Influence of sphingomyelin metabolism during epithelial-mesenchymal transition associated with aging in the renal papilla.

The renal collecting ducts (CD) are formed by a fully differentiated epithelium, and their tissue organization and function require the presence of mature cell adhesion structures. In certain circumstances, the cells can undergo de-differentiation by a process called epithelial-mesenchymal transition (EMT), in which the cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells, which includes loss of cell-cell adhesion. We have previously shown that in renal papillary CD cells, cell adhesion structures are located in sphingomyelin (SM)-enriched plasma membrane microdomains and the inhibition of SM synthase 1 activity induced CD cells to undergo an EMT process. In the present study, we evaluated the influence of SM metabolism during the EMT of the cells that form the CD of the renal papilla during aging. To this end, primary cultures of renal papillary CD cells from young, middle-, and aged-rats were performed. By combining biochemical and immunofluorescence studies, we found experimental evidence that CD cells undergo an increase in spontaneous and reversible EMT during aging and that at least one of the reasons for this phenomenon is the decrease in SM content due to the combination of decreased SM synthase activity and an increase in SM degradation mediated by neutral sphingomyelinase. Age is a risk factor for many diseases, among which renal fibrosis is included. Our findings highlight the importance of sphingolipids and particularly SM as a modulator of the fate of CD cells and probably contribute to the development of treatments to avoid or reverse renal fibrosis during aging.

Apoptotic cell extrusion depends on single-cell synthesis of sphingosine-1-phosphate by sphingosine kinase 2.

Collecting duct cells are physiologically subject to the hypertonic environment of the kidney. This condition is necessary for kidney maturation and function but represents a stress condition that requires active strategies to ensure epithelial integrity. Madin-Darby Canine Kidney (MDCK) cells develop the differentiated phenotype of collecting duct cells when subject to hypertonicity, serving as a model to study epithelial preservation and homeostasis in this particular environment. The integrity of epithelia is essential to achieve the required functional barrier. One of the mechanisms that ensure integrity is cell extrusion, a process initiated by sphingosine-1-phosphate (S1P) to remove dying or surplus cells while maintaining the epithelium barrier. Both types start with the activation of S1P receptor type 2, located in neighboring cells. In this work, we studied the effect of cell differentiation induced by hypertonicity on cell extrusion in MDCK cells, and we provide new insights into the associated molecular mechanism. We found that the different stages of differentiation influence the rate of apoptotic cell extrusion. Besides, we used a novel methodology to demonstrate that S1P increase in extruding cells of differentiated monolayers. These results show for first time that cell extrusion is triggered by the single-cell synthesis of S1P by sphingosine kinase 2 (SphK2), but not SphK1, of the extruding cell itself. Moreover, the inhibition or knockdown of SphK2 prevents cell extrusion and cell-cell junction protein degradation, but not apoptotic nuclear fragmentation. Thus, we propose SphK2 as the biochemical key to ensure the preservation of the epithelial barrier under hypertonic stress.

Sphingosine kinase and sphingosine-1-phosphate regulate epithelial cell architecture by the modulation of de novo sphingolipid synthesis.

Sphingolipids regulate several aspects of cell behavior and it has been demonstrated that cells adjust their sphingolipid metabolism in response to metabolic needs. Particularly, sphingosine-1-phosphate (S1P), a final product of sphingolipid metabolism, is a potent bioactive lipid involved in the regulation of various cellular processes, including cell proliferation, cell migration, actin cytoskeletal reorganization and cell adhesion. In previous work in rat renal papillae, we showed that sphingosine kinase (SK) expression and S1P levels are developmentally regulated and control de novo sphingolipid synthesis. The aim of the present study was to evaluate the participation of SK/S1P pathway in the triggering of cell differentiation by external hypertonicity. We found that hypertonicity evoked a sharp decrease in SK expression, thus activating the de novo sphingolipid synthesis pathway. Furthermore, the inhibition of SK activity evoked a relaxation of cell-cell adherens junction (AJ) with accumulation of the AJ complex (E-cadherin/ß-catenin/α-catenin) in the Golgi complex, preventing the acquisition of the differentiated cell phenotype. This phenotype alteration was a consequence of a sphingolipid misbalance with an increase in ceramide levels. Moreover, we found that SNAI1 and SNAI2 were located in the cell nucleus with impairment of cell differentiation induced by SK inhibition, a fact that is considered a biochemical marker of epithelial to mesenchymal transition. So, we suggest that the expression and activity of SK1, but not SK2, act as a control system, allowing epithelial cells to synchronize the various branches of sphingolipid metabolism for an adequate cell differentiation program.

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development.

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.

Bradykinin mediates the association of collecting duct cells to form migratory colonies, through B2 receptor activation.

It is known that bradykinin (BK) B2 receptor (B2R) is expressed in the collecting duct (CD) cells of the newborn rat kidney, but little is known about its role during early postnatal life. Therefore, we hypothesize that BK could participate in the mechanisms that mediate CD formation during the postnatal renal development. Performing primary cultures, combined with biochemical, immunocytochemical, and time-lapse analysis, we studied the role of BK in CD cell behavior isolated from renal papilla of neonatal rats. A reverse relationship was observed between B2R expression and the degree of CD epithelial cell sheet maturation. BK stimulation induced CD cell association upon B2R activation. The lack of B2R expression in cells showing mature adherens junctions suggested that BK is mostly involved in early adhesive events, thus favoring the initial formation of CD during development. Time-lapse analysis revealed that BK induced a high protrusive activity of CD cells, denoted by ruffle formation and lamellipodia extension. PI3K was involved in the BK-induced CD cell-cell association and the acquisition of the migratory phenotype since, when inhibited, membrane ruffles, and filopodia between cells diminished. Results indicate that the actions of BK mediated by PI3K activation were due to the downstream Akt and Rac pathways. This study, performed with CD cells that were not genetically manipulated, provides new experimental evidence supporting a novel role of BK in rat renal CD organization. As B2R blockade results in abnormal tubular differentiation, our results contribute to better understanding the etiology of human congenital renal malformation and diseases.

The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype.

Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.

Changes in ceramide metabolism are essential in Madin-Darby canine kidney cell differentiation.

Ceramides (Cers) and complex sphingolipids with defined acyl chain lengths play important roles in numerous cell processes. Six Cer synthase (CerS) isoenzymes (CerS1-6) are the key enzymes responsible for the production of the diversity of molecular species. In this study, we investigated the changes in sphingolipid metabolism during the differentiation of Madin-Darby canine kidney (MDCK) cells. By MALDI TOF TOF MS, we analyzed the molecular species of Cer, glucosylceramide (GlcCer), lactosylceramide (LacCer), and SM in nondifferentiated and differentiated cells (cultured under hypertonicity). The molecular species detected were the same, but cells subjected to hypertonicity presented higher levels of C24:1 Cer, C24:1 GlcCer, C24:1 SM, and C16:0 LacCer. Consistently with the molecular species, MDCK cells expressed CerS2, CerS4, and CerS6, but with no differences during cell differentiation. We next evaluated the different synthesis pathways with sphingolipid inhibitors and found that cells subjected to hypertonicity in the presence of amitriptyline, an inhibitor of acid sphingomyelinase, showed decreased radiolabeled incorporation in LacCer and cells did not develop a mature apical membrane. These results suggest that hypertonicity induces the endolysosomal degradation of SM, generating the Cer used as substrate for the synthesis of specific molecular species of glycosphingolipids that are essential for MDCK cell differentiation.

Participation of prostaglandin D2 in the mobilization of the nuclear-localized CTP:phosphocholine cytidylyltransferase alpha in renal epithelial cells.

Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location.

Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity.

Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and ß- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.

Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway.

In epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents.

Glycosphingolipid synthesis is essential for MDCK cell differentiation.

Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway.

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells.

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.

Potentiation of omega-3 fatty acid antidepressant-like effects with low non-antidepressant doses of fluoxetine and mirtazapine.

Despite the advances in psychopharmacology, the treatment of depressive disorders is still not satisfactory. Side effects and resistance to antidepressant drugs are the greatest complications during treatment. Based on recent evidence, omega-3 fatty acids may influence vulnerability and outcome in depressive disorders. The aim of this study was to further characterize the omega-3 antidepressant-like effect in rats in terms of its behavioral features in the depression model forced swimming test either alone or in combination with antidepressants fluoxetine or mirtazapine. Ultimately, we prompted to determine the lowest dose at which omega-3 fatty acids and antidepressant drugs may still represent a pharmacological advantage when employed in combined treatments. Chronic diet supplementation with omega-3 fatty acids produced concentration-dependent antidepressant-like effects in the forced swimming test displaying a behavioral profile similar to fluoxetine but different from mirtazapine. Fluoxetine or mirtazapine at antidepressant doses (10 and 20 mg/kg/day, respectively) rendered additive effects in combination with omega-3 fatty acid supplementation (720 mg/kg/day). Beneficial effects of combined treatment were also observed at sub-effective doses (1 mg/kg/day) of fluoxetine or mirtazapine, since in combination with omega-3 fatty acids (720 mg/kg/day), antidepressants potentiated omega-3 antidepressant-like effects. The antidepressant-like effects occurred in the absence of changes in brain phospholipid classes. The therapeutic approach of combining omega-3 fatty acids with low ineffective doses of antidepressants might represent benefits in the treatment of depression, especially in patients with depression resistant to conventional treatments and even may contribute to patient compliance by decreasing the magnitude of some antidepressant dose-dependent side effects.

The rate-limiting enzyme in phosphatidylcholine synthesis is associated with nuclear speckles under stress conditions.

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity.

The expression of sphingosine kinase-1 in head and neck carcinoma.

Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression.

Bradykinin induces formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2 in renal papillary collecting duct cells.

Focal adhesions (FAs) are structures of cell attachment to the extracellular matrix. We previously demonstrated that the intrarenal hormone bradykinin (BK) induces the restructuring of FAs in papillary collecting duct cells by dissipation of vinculin, but not talin, from FAs through a mechanism that involves PLCbeta activation, and that it also induces actin cytoskeleton reorganization. In the present study we investigated the mechanism by which BK induces the dissipation of vinculin-stained FAs in collecting duct cells. We found that BK induces the internalization of vinculin by a noncaveolar and independent pinocytic pathway and that at least a fraction of this protein is delivered to the recycling endosomal compartment, where it colocalizes with the transferrin receptor. Regarding the reassembly of vinculin-stained FAs, we found that BK induces the formation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-enriched vinculin-containing vesicles, which, by following a polarized exocytic route, transport vinculin to the site of FA assembly, an action that depends on actin filaments. The present study, which was carried out with cells that were not genetically manipulated, shows for the first time that BK induces the formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2, which transport vinculin to the site of FA assembly. Therefore, the modulation of the formation of these vesicle-like structures could be a physiological mechanism through which the cell can reuse the BK-induced internalized vinculin to be delivered for newly forming FAs in renal papillary collecting duct cells.

Expression and activity of SGLT2 in diabetes induced by streptozotocin: relationship with the lipid environment.

BACKGROUND/AIMS: Diabetes mellitus may impact on the regulation of renal Na+-glucose cotransporter type 2 (SGLT2), however, previous studies have yielded conflicting results on the effects of streptozotocin (STZ)-induced diabetes on SGLT-mediated glucose transport. METHODS: Diabetes was induced in male Wistar rats. The studies were performed at 3 (D3), 7 (D7) and 14 (D14) days after a single i.p. injection of STZ. SGLT2 activity was measured using alpha-14C-methyl glucose uptake in brush-border vesicles (BBV) from renal cortex, and SGLT2 expression was assessed by immunoblotting. Phospholipids were quantified by a modification of Fiske-Subarow's method after being separated by thin-layer chromatography. RESULTS: Glucose uptake was reduced in all groups of diabetic rats. SGLT2 expression decreased in D3 and D7. There was a decrease in sphingomyelin (SM) content and an increase in phosphatidylcholine (PC) content in BBV from D14 versus control, without differences in phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylethanolamine (PE). CONCLUSION: The downregulation of SGLT2 activity during STZ-induced diabetes may be a protective mechanism to control the excess of circulating glucose and could be a consequence of a decrease in SGLT2 expression in D3 and D7, whereas altered activity of SGLT2 in D14 could be a consequence of changes in membrane lipid composition. However, we cannot discard the possibility that the decrease in SGLT2 activity could be due to a covalent modification of the active site of the protein.

Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells.

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.