Gamba, Gerardo

Slc26a9 is an electrogenic n Cl − /HCO 3 − exchanger with a large anion conductance that is mainly expressed in respiratory and gastrointestinal epithelial cells. It was recently shown that Slc26a9 is negatively regulated by the serine/threonine kinases WNK1, WNK3, and WNK4, and that these effects were not dependent upon the kinase activity. However, WNK3 and WNK4 have been shown to exhibit opposite effects in other chloride cotransporters. To gain insight to putative kinase regulation of Slc26a9, we evaluated the effect of protein kinases WNK3 and WNK4 on Slc26a9‐mediated anion transport using 36 Cl − uptake in Xenopus laevis oocytes. Oocytes were injected with Slc26a9‐cRNA alone or together with WNK4 or WNK3‐cRNA. We observed that WNK4 caused a ~30% decrease in 36 Cl − uptake by Slc26a9. This inhibition was not observed with co‐injection of D318A‐WNK4 which renders WNK4 catalytically inactive. Co‐injection of oocytes with Slc26a9 and WNK3 increased Slc26a9 activity by ~40% above baseline. This increased Slc26a9 activity by WNK3 was also kinase dependent, i.e., Slc26a9 activation was eliminated by D294A‐WNK3 which renders WNK3 catalytically inactive. Thus, our data indicate that WNK3 is a positive regulator of transport Cl − and WNK4 is a negative regulator of Cl − transport by the Slc26a9 anion transporter/channel. Furthermore, this Slc26a9 transport regulation is dependent on kinase activity of WNK3 and WNK4.

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)

The electroneutral (zSMCTn/Slc5a12) and electrogenic (zSMCTe/Slc5a8) Na + /monocarboxylate (MC − ) transporters of zebrafish, move short chain fatty acids, ketone‐bodies, lactate, pyruvate and nicotinate but with clear differences in Na + : MC − stoichiometry and affinity. The affinity of zSMCTn for pyruvate, lactate and nicotinate is lower than zSMCTe (~6, ~10 and ~40 times respectively). It is known that valproate and nonsteroidal anti‐inflammatory drugs, such as ibuprofen, valproate reduce the activity of mammalian SMCTe, but this effect has not been characterized for SMCTn. Using 22 Na + uptake in Xenopus oocytes injected with either zSMCT's or hSMCT cRNA, we evaluated the kinetics of Na + propionate cotransport by SMCT's (hSMCTe, zSMCTe, zSMCTn). We also determined cotransport inhibition by ibuprofen, ketoprofen, naproxen and valproate of these oocytes in presence or absence of propionate. Our results indicate that zSMCTn (Km =830±110μM) has only 3 times less affinity for propionate than zSMCTe (Km =240±50μM) and hSMCTe (Km =200±20μM). Ibuprofen, ketoprofen, naproxen and valproate cause a significant reduction in 22 Na + uptake for zSMCTe, hSMCTe as well as zSMCTn in presence of propionate. Ibuprofen inhibition is more potent for zSMCTn (K i ~200μM) than zSMCTe (K i ~600μM) and hSMCTe (K i ~800μM). These functional and inhibitory differences may help define the binding site for MC − and Na + of SMCT proteins.

The Na + /monocarboxylate transporters (SMCT1 and SMCT2) are expressed several organ between them intestine and kidney, suggesting that its main physiological function are the absorption of fuel molecules involved in cellular metabolism and have only one ‐COOH group in its chemical structure; (lactate, propionate). Drugs with similar chemical properties also are translocated by SMCTs (B3 vitamin, pyrazinoate). However some propionate‐derivative NSAIDs (ibuprofen) reduce the activity of mammalian SMCT1. Using 22 Na + uptake in Xenopus oocytes injected with zebrafish SMCT’s (zSMCT1, zSMCT2) or human SMCT1 cRNA, we evaluated the effect of aspirin and salicylate on the function SMCT’s. Our results indicate a 22 Na + uptake that is aspirin concentration dependent in all SMCT’s. Similar affinity by aspirin was observed between hSMCT1 (K m ~2.4mM) and zSMCT1 (K m ~2.5 mM) in contrast to a lower affinity of zSMCT2 (K m ~5mM). Also the difference of substrate affinity between zSMCT2 and hSMCT1 was evident by salicylate ( hSMCT1 K m ~4mM vs zSMCT1 K m ~30 mM). Interestingly, although zSMCT1 and hSMCT1 shares a similar affinity and transport capacity for several substrates, the salicylate transport by zSMCT1 was not detected and only reduced a small percentage propionate transport induced by zSMCT1 (~10%) in contrast of ibuprofen which drops to 80%. The clear difference of salicylate transporter between SMCT1 could be explained by changes in the structure of proteins because the amino acids sequences are approximately 50% different, so the salicylate could be candidate for studies of structural‐function between SMCT1. Grant Funding Source : CONACYT48709MJ(CP)

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.