Protein kinase C mediated intracellular signaling pathways are involved in the regulation of sodium-dependent glucose co-transporter SGLT1 activity.

The sodium-dependent glucose co-transporter (SGLT1) is regulated by protein kinases. The aim of the present study was to examine the role of protein kinase C (PKC) in the regulation of rabbit (rb) SGLT1 activity as determined by alpha-methyl-D-glucopyranoside (AMG) uptake and to identify the cellular mechanisms involved in this process. For this purpose Chinese hamster ovary cells expressing rbSGLT1 (CHO-G6D3) were treated with PKC activators and inhibitors. PKC activators did not exert any effect on AMG uptake, as corroborated by mutation of the putative phosphorylation sites of PKC. In contrast, the PKC inhibitor bisindolylmaleimide I (BIM) increased AMG uptake. This effect was associated with translocation of rbSGLT1 from the intracellular pool to the plasma membrane demonstrated by pre-treatment of G6D3 cells with cytochalasin D that abolished the effect of BIM. In addition, intracellular signaling pathways (p38/MAPK, ERK/MAPK, JNK/MAPK, and PI3K/Akt/mTOR) were associated with PKC-regulated AMG uptake. Moreover, rbSGLT1 mRNA level was higher in BIM-treated cells than in untreated, control cells. This effect was completely abolished by actinomycin D treatment. The present study demonstrates that PKC regulates rbSGLT1 activity via a complex intracellular mechanism that involves sorting and transcriptional regulation of rbSGLT1. The study findings suggest the involvement of two complementary opposite mechanism of action, in which the balance between two antagonistic effects, namely stimulation and inhibition of the transporter, regulates the activity of rbSGLT1 by PKC.

Protein kinase-A affects sorting and conformation of the sodium-dependent glucose co-transporter SGLT1.

In Chinese hamster ovary cells expressing rabbit sodium-dependent glucose transporter (rbSGLT1) protein kinase A (PKA) activators (forskolin and 8-Br-cAMP) stimulated alpha-methyl D-glucopyranoside uptake. Kinetic analysis revealed an increase in both V(max) and affinity of the transport. Immunohistochemistry and biotinylation experiments showed that this stimulation was accompanied by an increased amount of SGLT1 localized into the plasma membrane, which explains the higher V(max) of the transport. Cytochalasin D only partly attenuated the effect of forskolin as did deletion of the PKA phosphorylation site of SGLT1 in transient transfection studies. Experiments using an anti-phosphopeptide antibody revealed that forskolin also increased the extent of phosphorylation of SGLT1 in the membrane fraction. These results suggested that regulation of SGLT1 mediated glucose transport involves an additional direct effect on SGLT1 by phosphorylation. To evaluate this assumption further, phosphorylation studies of recombinant human SGLT1 (hSGLT1) in vitro were performed. In the presence of the catalytic subunit PKA and [(32)P] ATP 1.05 mol of phosphate were incorporated/mol of hSGLT1. Additionally, phosphorylated hSGLT1 demonstrated a reduction in tryptophan fluorescence intensity and a higher quenching by the hydrophilic Trp quencher acrylamide, particularly in the presence of D-glucose. These results indicate that PKA-mediated phosphorylation of SGLT1 changes the conformation of the empty carrier and the glucose carrier complex, probably causing the increase in transport affinity. Thus, PKA-mediated phosphorylation of the transporter represents a further mechanism in the regulation of SGLT1-mediated glucose transport in epithelial cells, in addition to a change in surface membrane expression.

Long-term smoking and ethanol exposure accentuates oxidative stress in hearts of mice.

The present study was undertaken to investigate the influence of ethanol on the cardiac antioxidant defense system of mice exposed to cigarette smoke. Cigarette smoke exposure for 10 wk led to an increase in the levels of lipid peroxidation in the heart. These levels increased further in animals co-exposed to ethanol and cigarette smoke. Concomitantly, the levels of glutathione were found to decrease after cigarette smoke exposure. In the animals co-exposed to ethanol and cigarette smoke, there was a further decrease in glutathione levels. Catalase activity increased in the animals exposed to cigarette smoke; this activity increased further with combined exposure to cigarette smoke and ethanol. On the other hand, superoxide dismutase (SOD) activity was not affected by cigarette smoke exposure, while it increased in the ethanol-exposed group. SOD activity was higher in the hearts of animals co-exposed to cigarette smoke and ethanol as compared to animals that received ethanol alone. The activity of heat-stable lactate dehydrogenase in serum was not affected when the animals were exposed to either cigarette smoke or ethanol, whereas this activity was observed to be elevated in animals co-exposed to cigarette smoke and ethanol. It appears from these results that ethanol potentates the cigarette-smoke-induced peroxidative damage to heart.