Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sodium-dependent glucose transporter SGLT1 is expressed on the apical plasma membrane of fully differentiated enterocytes. Recently, we have found that the cotransport function appears gradually during the process of differentiation of the human intestinal epithelial cell clone HT-29-D4. However, the SGLT1 protein was detected in both undifferentiated and differentiated HT-29-D4 cells suggesting that sodium-glucose cotransport was dependent on post-translational events controlling the efficient targeting of the protein in the plasma membrane. In the present study, we have analyzed the molecular mechanisms controlling the functional expression of the SGLT1 protein during the course of HT-29-D4 differentiation. We show that the appearance of the cotransport function in the apical membrane is blocked by 1-5-isoquinolinesulfonyl)-2-methylpiperazine-HCl (H-7), a potent inhibitor of protein kinase C activity. Moreover, H-7 treatment was associated with an inability of HT-29-D4 cells to organize into a polarized monolayer of differentiated cells. Reciprocally, short term treatment (15 min) of undifferentiated cells by 0.1 microM phorbol myristyl acetate resulted in the appearance of the cotransport function. In contrast, inhibition of cAMP and cGMP-dependent protein kinases by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl did not prevent the development of sodium-glucose cotransport during the differentiation of HT-29-D4 cells. In addition, stimulation of cAMP-dependent protein kinases by 8-Cl-cAMP did not induce the cotransport function in undifferentiated HT-29-D4 cells. By using immunogold labeling at the electron microscopy level, we demonstrated that phorbol myristyl acetate induced the redistribution of SGLT1 protein from intracellular sites to the plasma membrane. In conclusion, our data show that the appearance of a functional sodium-glucose cotransporter in HT-29-D4 cells is controlled, at least in part, by intracellular pathways regulated by the activity of protein kinase C.
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PMID:The development of Na(+)-dependent glucose transport during differentiation of an intestinal epithelial cell clone is regulated by protein kinase C. 775 99

We examined changes in the mRNA level of SGLT1, a Na+/glucose cotransporter, by the differentiation status of LLC-PK1 renal epithelial cells. Proliferating (undifferentiated) cells revealed no detectable SGLT1 mRNA by Northern blot analysis. However, when cells became confluent and differentiated into polarized monolayers, there was an abrupt appearance of the SGLT1 mRNA. When confluent (differentiated) cells were dedifferentiated by reseeding at a subconfluent density, SGLT1 mRNA levels decreased quickly to nondetectable levels (t1/2 = 1.5 h), while the mRNA levels of gamma-glutamyltranspeptidase, another differentiation marker, decreased only slowly (t1/2 > 40 h). This decrease in SGLT1 mRNA was completely blocked by H-7, a protein kinase inhibitor. Since protein kinase C was highly activated in the undifferentiated cells and treatment of differentiated cells with a phorbol ester also induced quick and complete loss of SGLT1 mRNA (t1/2 = 1.5 h) but not of gamma-glutamyltranspeptidase mRNA, protein kinase C activation appears to be involved in the dedifferentiation-induced decrease in SGLT1 mRNA. Although the phorbol ester-induced decrease in the SGLT1 mRNA level was blocked completely by inhibition of transcription, inhibitors of translation blocked the decrease in mRNA levels only partially.
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PMID:Differentiation-dependent expression of the Na+/glucose cotransporter (SGLT1) in LLC-PK1 cells: role of protein kinase C activation and ongoing transcription. 799 57

Cotransporters are proteins responsible for the accumulation of nutrients, neurotransmitters, and drugs in cells. As forskolin has been shown to stimulate intestinal Na+/glucose cotransport, we have used electrophysiological techniques to examine the role of protein kinases in regulating Na+/glucose cotransporters, SGLT1, expressed in Xenopus laevis oocytes. We monitored SGLT1 kinetics, the number of SGLT1 cotransporters in the plasma membrane, and plasma membrane area before and after activation of protein kinases. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and sn-1, 2-dioctanoylglycerol (DOG) were used as membrane permeable activators of protein kinases A (PKA) and C (PKC), respectively. In oocytes expressing rabbit SGLT1 8-Br-cAMP increased by 28 +/- 4% (n = 10), and DOG decreased by 51 +/- 5% (n = 13) the maximum rate of Na+/glucose cotransport. These reversible changes in the maximum transport rate occurred within minutes, and were accompanied by proportional changes in the number of cotransporters in the membrane and area of the plasma membrane. This suggests that protein kinases regulate rabbit SGLT1 activity by controlling the distribution of transporters between intracellular compartments and the plasma membrane, and that this occurs by exo- and endocytosis. Similar increases in maximum transport were obtained with activation of PKA in oocytes expressing rabbit, human, and rat SGLT1 isoforms, but with activation of PKC the response was isoform-dependent. PKC activation decreased the maximum rate of transport by rabbit and rat SGLT1, but increased transport by human SGLT1. We conclude that: (i) the regulation of SGLT1 expression in oocytes by protein kinases occurs mainly by regulated endo- and exocytosis; (ii) it is independent of consensus phosphorylation sites in the transporter; and (iii) the effect of a given kinase depends upon the actual sequence of the cotransporter expressed. These considerations may also apply to the regulation of other cotransporters by protein kinases in oocytes, cells, and tissues.
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PMID:Regulation of Na+/glucose cotransporter expression by protein kinases in Xenopus laevis oocytes. 866 46

Na+/glucose cotransporters (SGLTs) are expressed in the small intestine and the proximal renal tubule, where they play a central role in the absorption of glucose and galactose from food and the reabsorption of glucose from the glomerular filtrate. The regulation of intestinal sugar absorption occurs over two distinct time scales, one over days and the other over minutes. This review focuses on the mechanisms involved in the shorter-term regulation. Recent studies of the mouse intestine in vitro demonstrated that Na+/glucose cotransport is increased two- to eightfold within minutes by the application of forskolin, an agent that increases intracellular cyclic AMP levels. Here we explore how cyclic AMP may upregulate Na+/glucose cotransport. Our strategy was to express cloned SGLT1s in Xenopus laevis oocytes and then use electrophysiological methods to measure (i) the kinetics of Na+/glucose cotransport, (ii) the number of cotransporters in the plasma membrane, and (iii) the net rate of exo- and endocytosis before and after activation of protein kinases. To evaluate the role of cotransporter phosphorylation, we have examined the effect of protein kinase activation on various SGLT1 isoforms and other cotransporters. In oocytes expressing rabbit SGLT1, the activation of protein kinase A (PKA) increased the maximum rate of Na+/glucose cotransport by 30%, and the activation of protein kinase C (PKC) decreased the maximum rate of transport by 60%. Changes in maximum transport rates were accompanied by proportional changes in the number of cotransporters in the plasma membrane and by changes in the area of the membrane. We conclude that PKA and PKC regulate rabbit SGLT1 activity by modulating the number of cotransporters in the plasma membrane and that this occurs through regulation of exocytosis and endocytosis. Given the size of intracellular transport vesicles containing SGLT1, 100-120 nm in diameter, and the density of cotransporters in these vesicles, 10-20 per vesicle, we estimate that the net rate of SGLT1 vesicle exocytosis is about 10,000 s-1 and that this rate increases 100-fold after activation of PKA. The effect of PKA is independent of the presence or absence of consensus sites for phosphorylation on SGLT1. Surprisingly, the effects of PKA or PKC depend critically on the sequence of the contransporter being expressed in the oocyte, e.g. activation of PKC inhibited rabbit and rat SGLT1, but stimulated human SGLT1. This dependency suggests that the regulation of vesicle trafficking by protein kinases depends upon the structure of the cotransporter expressed in the oocyte. Similar considerations apply to other classes of cotransporters, such as the neurotransmitter and dipeptide cotransporters. Our working hypothesis is that the regulation of cotransporter expression by protein kinases occurs largely by regulated exo- and endocytosis, and that the effect of the protein kinases is indirect and determined by critical domains in the cotransporter.
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PMID:Regulation of Na+/glucose cotransporters. 905 Feb 36

Several glucose transporters have recently been identified in glomeruli, and in cultured glomerular cells. These include the facilitative glucose transporter isoforms GLUTs 1, 3 and 4, and sodium-glucose cotransport activity with characteristics of SGLT1. GLUTs 1, 3 and 4 are all high affinity, low capacity, facilitative glucose transporters which typically would be saturated at or near physiologic glucose concentrations. The SGLT transporter of mesangial cells is also a high affinity transporter which similarly could be saturated under normal glucose conditions. This suggests that in order for mesangial cells to take up excessive quantities of glucose in diabetes, changes in glucose transporter expression, translocation or activity may be required. Accordingly, recent investigations discovered positive-feedback regulation of the mesangial cell GLUT1 transporter by glucose, and a regulatory role for GLUT1 in glucose metabolism and extracellular matrix synthesis. Future investigations of glucose transporters in the pathogenesis of diabetic renal disease will now likely proceed in multiple directions, including but not limited to: (1) examination of their regulation by growth factors implicated in diabetic nephropathy, and the resultant effects on ECM synthesis; (2) determination of the mechanisms by which GLUT1 regulates the expression of aldose reductase, PKC, GLUT1, and other genes in the mesangial cell; and (3) Suppression of glucose transporters in attempts to prevent high glucose-induced diabetic glomerulosclerosis.
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PMID:Glucose transporters of the glomerulus and the implications for diabetic nephropathy. 928 9

We have used the recombinant NH2-terminal myc-tagged rabbit Na+-glucose transporter (SGLT1) to study the regulation of this carrier expressed in COS-7 cells. Treatment of cells with a protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), caused a significant decrease (38.03 +/- 0.05%) in methyl alpha-D-glucopyranoside transport activity that could not be emulated by 4alpha-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKC inhibitor bisindolylmaleimide I. The maximal rate of Na+-glucose cotransport activity (Vmax) was decreased from 1.29 +/- 0.09 to 0.85 +/- 0.04 nmol. min-1. mg protein-1 after PMA exposure. However, measurement of high-affinity Na+-dependent phloridzin binding revealed that there was no difference in the number of cell surface transporters after PMA treatment; maximal binding capacities were 1.54 +/- 0.34 and 1.64 +/- 0.21 pmol/mg protein for untreated and treated cells, respectively. The apparent sugar binding affinity (Michaelis-Menten constant) and phloridzin binding affinity (dissociation constant) were not affected by PMA. Because PKC reduced Vmax without affecting the number of cell surface SGLT1 transporters, we conclude that PKC has a direct effect on the carrier, resulting in a lowering of the transporter turnover rate by a factor of two.
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PMID:PKC regulates turnover rate of rabbit intestinal Na+-glucose transporter expressed in COS-7 cells. 1032 52

Perfusion of rat jejunum in vitro with PMA increased fructose transport by 70% compared with control values and was blocked by the protein kinase C (PKC) inhibitor chelerythrine. The brush-border membrane contained both the fructose transporters GLUT5 and GLUT2; the presence of the latter was confirmed by luminal biotinylation. PMA increased the GLUT2 level 4-fold within minutes, so that the level was comparable with that of the basolateral membrane, but had no effect on GLUT5 level. GLUT2 was functional, accessible to luminal fructose and could be inhibited selectively by phloretin to permit determination of GLUT2- and GLUT5-mediated transport components. The 4-fold increase in GLUT2 level induced by PMA was matched by a 4-fold increase in GLUT2-mediated transport: there was a compensatory fall in the GLUT5-mediated rate. The pattern of dynamic trafficking was seen only for GLUT2, not GLUT5 or SGLT1, implying that GLUT2 trafficks to the brush-border membrane by a different pathway. Trafficking of GLUT2 to the brush-border membrane correlated with activation of PKC betaII, implying that this isoenzyme is likely to control trafficking. Since PKC is activated by endogenous hormones, GLUT2 levels in vivo are 3-4-fold those in vitro; moreover, because PKC is inactivated as soon as intestine is excised, GLUT2 is lost from the brush-border within minutes in vitro. It is therefore difficult to detect GLUT2 in most in vitro preparations and its role in intestinal sugar absorption across the brush-border membrane has accordingly been overlooked.
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PMID:Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C. 1092 38

We have investigated the mechanism responsible for the diffusive component of intestinal glucose absorption, the major route by which glucose is absorbed. In perfused rat jejunum in vivo, absorption was strongly inhibited by phloretin, an inhibitor of GLUT2. The GLUT2 level at the brush-border membrane increased some 2-fold when the luminal glucose concentration was changed from 0 to 100 mM. The phloretin-sensitive or diffusive component of absorption appeared superficially linear and consistent with simple diffusion, but was in fact carrier-mediated and co-operative (n=1.6, [G(1/2)]=56 mM; where [G(1/2)] is the glucose concentration at half V(max)) because of the glucose-induced activation and recruitment of GLUT2 to the brush-border membrane. Diffusive transport by paracellular flow was negligible. The phloretin-insensitive, SGLT1-mediated, component of glucose absorption showed simple saturation kinetics with [G(1/2)]=27 mM: the activation of protein kinase C (PKC) betaII, the isoenzyme of PKC that most probably controls GLUT2 trafficking [Helliwell, Richardson, Affleck and Kellett (2000) Biochem. J. 350, 149-154], also showed simple saturation kinetics, with [G(1/2)]=21 mM. We conclude that the principal route for glucose absorption is by GLUT2-mediated facilitated diffusion across the brush-border membrane, which is up to 3-fold greater than that by SGLT1; the magnitude of the diffusive component at any given glucose concentration correlates with the SGLT1-dependent activation of PKC betaII. The implications of these findings for the assimilation of sugars immediately after a meal are discussed.
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PMID:The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. 1092 39

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.
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PMID:Differential regulation of three glucose transporter genes in a renal epithelial cell line. 1102 46

Over the last decade, a debate has developed about the mechanism of the passive or 'diffusive' component of intestinal glucose absorption and, indeed, whether it even exists. Pappenheimer and colleagues have proposed that paracellular solvent drag contributes a passive component, which, at high concentrations of sugars similar to those in the jejunal lumen immediately after a meal, is severalfold greater than the active component mediated by the Na+-glucose cotransporter SGLT1. On the other hand, Ferraris & Diamond maintain that the kinetics of glucose absorption can be explained solely in terms of SGLT1 and that a passive or paracellular component plays little, if any, part. Recently, we have provided new evidence that the passive component of glucose absorption exists, but is in fact facilitated since it is mediated by the rapid, glucose-dependent activation and recruitment of the facilitative glucose transporter GLUT2 to the brush-border membrane; regulation involves a protein kinase C (PKC)-dependent pathway activated by glucose transport through SGLT1 and also involves mitogen-activated protein kinase (MAP kinase) signalling pathways. This topical review seeks to highlight the significant points of the debate, to show how our proposals on GLUT2 impact on different aspects of the debate and to look at the regulatory events that are likely to be involved in the short-term regulation of sugar absorption during the assimilation of a meal.
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PMID:The facilitated component of intestinal glucose absorption. 1125 Oct 42


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