Expression of the Na؉ -coupled glucose cotransporter SGLT1 is regulated post-transcriptionally at the level of mRNA stability. We have previously demonstrated that cAMP-dependent stabilization of the SGLT1 message was correlated with the protein phosphorylationdependent binding of cytoplasmic proteins to a uridinerich sequence (URE) in the 3-untranslated region (UTR). In the present study, the regulatory role of the URE was demonstrated by inserting it into the 3-UTR of a ␤-globin reporter minigene under the control of the tetracycline-regulated promoter. The resultant chimeric globin/SGLT1 mRNA expressed after transfection into LLC-PK1 cells exhibited a decreased half-life compared with the ␤-globin control, indicating that the URE serves a destabilizing function. Activation of protein kinase A stabilized the chimeric message but not the ␤-globin control, indicating the presence of a regulatory stabilizing sequence within the URE. A 38-kDa nucleocytoplasmic protein was identified that recognized a 12-nucleotide binding site within the URE. A mutation in this binding site that prevented protein binding assayed in vitro by UV cross-linking also prevented protein kinase Adependent stabilization of the chimeric message assayed in vivo. These findings identify the interaction between a 38-kDa nucleocytoplasmic protein and a regulatory uridinerich sequence in the 3-UTR as critical for cAMP-mediated SGLT1 message stabilization.Glucose is the major carbon and energy source for eukaryotic cells. Transport of glucose into mammalian cells is the ratelimiting step for its utilization and accordingly is a highly regulated process. Maintenance of a constant blood glucose level is essential for cellular homeostasis. In kidney, glucose is reabsorbed from the urinary filtrate by the action of several types of glucose transporters arranged in series along the proximal tubule. These function together in polarized epithelial cells to mediate transepithelial transport of glucose. SGLT 1 (Na ϩ /glucose) cotransporters in the apical membrane catalyze active glucose transport into the cell driven by the Na ϩ gradient (1). Glucose diffuses passively out of the cell into the circulation via basolateral GLUT facilitative transporters (2). High affinity glucose transporters SGLT1 and GLUT1 located in late proximal tubule scavenge the remainder of filtered glucose not reabsorbed in early proximal tubule (3). SGLT1 is also expressed in small intestine where it mediates absorption of dietary glucose and galactose (3, 4).Most of our current information concerning the regulation of SGLT1 expression has been obtained from studies using the polarized epithelial cell line LLC-PK 1 , derived from porcine kidney (5). SGLT1 is expressed in this cell line as shown by cDNA cloning and Northern blot analysis (6) and is highly regulated by the cell growth and differentiation state (5,7,8). Protein kinase A (PKA) and protein kinase C (PKC) exert opposing effects on SGLT1 mRNA stability and steady-state levels in confluent cultures. Activation of PKA resu...

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The porcine kidney epithelial cell line LLC-PK1 expresses a sodium-coupled glucose cotransporter (SGLT1) together with other differentiation markers of renal proximal tubule such as trehalase and gamma-glutamyltranspeptidase. Expression is regulated by cell density and exogenous differentiation inducers such as hexamethylene bisacetamide (HMBA). Northern blot and PCR analysis of clonal cell populations indicated SGLT1 mRNA was not detectable in subconfluent cultures, but 2.2 and 3.9 kb SGLT1 mRNA species appeared after cell confluence, accompanying expression of the transport activity. SGLT1 mRNA levels were significantly increased after treatment of confluent cultures with HMBA, paralleling increases in the transport activity and immunodetectable 75 kD cotransporter subunit. SGLT1 mRNA was also increased after treatment of cultures with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), an inducer of Na+/glucose cotransport activity. The 3.9 kb SGLT1 transcript showed the largest increase after either HMBA or IBMX treatment. HMBA treatment also resulted in increased mRNA levels of two other differentiation markers--trehalase and gamma-glutamyltranspeptidase. By contrast, trehalase and gamma-glutamyltranspeptidase mRNA levels were not increased by IBMX. Regulation of Na+/glucose symporter expression by either cell density, cyclic AMP elevation, or differentiation inducer treatment occurs, at least in part, at the level of SGLT1 mRNA and can be dissociated from regulation of other differentiation markers.