Mount, David B.

SMCTe (SLC5A8) transports several monocarboxylates, including short‐chain fatty acids, ketone‐bodies, pyruvate, lactate, and keto‐isocaproic acid (KIC). We found SMCTe mRNA in zebrafish pancreas ( in situ hybridization); other groups have demonstrated that SLC5A8 gene silencing is associated with pancreatic cancer. Extracellular KIC stimulates insulin secretion in pancreas, suggesting a potential role for SMCTe. However, the physiological role of SMCTe in pancreas is unknown. Using RT‐PCR with rat SMCTe‐specific primers, we found that SMCTe mRNA is expressed in rat pancreas. Using Western Blot analysis with specific SMCTe antibodies, we also detected SMCTe protein in rat pancreas (~75 kDa). We next tested the effect of insulin on SMCTe function using 22 Na + uptake in Xenopus oocytes injected with human SMCTe (hSMCTe) or co‐injected with hSGK1 cRNA. Specifically, we evaluated transport 22 Na + uptake elicited by 40 U insulin in presence or absence of KIC. Insulin reduced KIC‐mediated 22 Na + uptake transport by ~25% in hSMCTe‐oocytes. Human SGK1 also reduced KIC‐mediated 22 Na + uptake transport by ~25% in hSMCTe‐oocytes. Co‐expression of hSMCTe and hSGK1 in oocytes did not further reduce KIC‐mediated 22 Na + uptake. These results indicate that the signal transduction pathway by which insulin and SKG1 reduce SMCTe transport activity overlaps. CONACYT48709MJ(CP), EY017732 (MFR), DK070756 (DM), DK64635(GG)

Many diseases are caused by altered epithelial Cl − or HCO 3 − transport. Mutations in the SLC26A3 transporter are causative for congenital chloride‐losing diarrhea; however, no other SLC26 transporter plays such a direct genetic role in disorders of epithelial Cl − transport. SLC26A9 is expressed in the lung, stomach, intestine and kidney (lesser extent) but its physiological function in these epithelia is not clear. Thus far, no diseases result from SLC26A9 mutations. However, database analysis reveals three single nucleotide polymorphisms (SNPs). We reasoned that the human SLC26A9‐SNPs would provide insight into what a disease phenotype might be functionally manifest. Here we analyzed the ion transport characteristics of human SLC26A9 and its SNPs: V622L, V744M and H748R. Human SLC26A9 and its three SNPs were expressed in Xenopus laevis oocytes. Effects on ion transport were evaluated using ion selective electrodes and voltage clamping. Measuring Cl − and HCO 3 − flux, our results show that SLC26A9 is a Cl − /HCO 3 − exchanger. One SNP, V622L, is located in the C‐terminus just before the STAS domain of SLC26A9. V622L has ~50% function (current and Cl − transport). These results suggest that the possession of V622L allele in humans would decrease SLC26A9 function and modulate the risk for lung disease (cystic fibrosis), gut disease (gastric ulcer) or hypertension, due to reduced Cl − transport across epithelial cells.

The ten‐member SLC26 gene family encodes anion transporters with a diversity of substrates and transport modes, functioning as electrogenic exchangers, anion sensors, and/or anion channels. Slc26a6 is perhaps the most versatile paralog, mediating electrogenic exchange of multiple divalent and monovalent anions. We have generated a cysteineless Slc26a6 for structure‐function studies, fully substituting 8 cysteines with threonine, serine, and alanine, as dictated by the functional data for single and compound mutants. Although absolute transport rates are reduced in the cysteineless Slc26a6 protein it exhibits equivalent anion specificity and electrophysiology (chloride‐bicarbonate exchange) to that of wild‐type. On Western blots, the oligomerization of wild‐type Slc26a6 is inhibited by dithiothreitol, suggesting a role for inter‐cysteine disulfide bonds; oligomerization is absent in the cysteineless protein. Co‐immunoprecipitation of FLAG‐ and MYC‐tagged Slc26a6 is easily detectable for wild‐type proteins but absent in the epitope‐tagged cysteineless proteins. The equivalent data for single and compound mutants implicate C‐terminal cytoplasmic cysteines in oligomerization of the transporter protein. In summary, the eight cysteines in Slc26a6 are dispensable for transport activity but appear to play a role in oligomerization of the transport protein.

SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the coinjection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the coinjection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 coinjection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis, and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.