Potential role of the ABCG2-Q141K polymorphism in type 2 diabetes

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease and variations in multispecific membrane transporter functions may affect T2DM development, complications or treatment. In this work we have analyzed the potential effects of a major polymorphism, the Q141K variant of the ABCG2 transporter in T2DM. The ABCG2 protein is a multispecific xeno- and endobiotic transporter, affecting drug metabolism and playing a key role in uric acid extrusion. The ABCG2-Q141K variant, with reduced expression level and function, is present in 15–35% of individuals, depending on the genetic background of the population, and has been shown to significantly affect gout development. Several other diseases, including hypertension, chronic renal failure, and T2DM have also been reported to be associated with high serum uric acid levels, suggesting that ABCG2 may also play a role in these conditions. In this work we have compared relatively small cohorts (n = 203) of T2DM patients (n = 99) and healthy (n = 104) individuals regarding the major laboratory indicators of T2DM and determined the presence of the SNP rs2231142 (C421A), resulting the ABCG2-Q141K protein variant. We found significantly higher blood glucose and HbA1c levels in the T2DM patients carrying the ABCG2-Q141K variant. These findings may emphasize the potential metabolic role of ABCG2 in T2DM and indicate that further research should explore how prevention and treatment of this disease may be affected by the frequent polymorphism of ABCG2.

Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3--cotransport and Na+/H+-exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+-exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3--cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+-exchanger NHE1/SLC9A1 and Na+,HCO3--cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3--cotransport associates with severe breast cancer: enlarged CO2/HCO3--dependent rises in pHi predict accelerated cell proliferation; whereas enhanced CO2/HCO3--dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from Luminal A or Basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3--cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.

Changing Wheat Bran Structural Properties by Extrusion-Cooking on a Pilot and Industrial Scale: A Comparative Study

Extrusion-cooking can be used to change the techno–functional and nutrition-related properties of wheat bran. In this study, pilot-scale (BC21) and industrial-scale (BC45) twin-screw extrusion-cooking using different types of extrusion (single-pass, double-pass and acid extrusion-cooking) and process parameters (temperature, moisture) were compared for their impact on wheat bran. When applying the same process settings, the higher strong water-binding capacity, extract viscosity and extractability displayed by bran extruded using the industrial set-up reflected a more considerable wheat bran structure degradation compared to pilot-scale extrusion-cooking. This was attributed to the overall higher specific mechanical energy (SME), pressure and product temperature that were reached inside the industrial extruder. When changing the type of extrusion-cooking from single-pass to double-pass and acid extrusion-cooking, wheat bran physicochemical characteristics evolved in the same direction, irrespective of extruder scale. The differences in bran characteristics were, however, smaller on industrial-scale. Results show that the differentiating power of the latter can be increased by decreasing the moisture content and increasing product temperature, beyond what is possible in the pilot-scale extruder. This was confirmed by using a BC72 industrial-scale extruder at low moisture content. In conclusion, the extruder scale mainly determines the SME that can be reached and determines the potential to modify wheat bran.

The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations

Atomic-level structural insight on the human ABCG2 membrane protein, a pharmacologically important transporter, has been recently revealed by several key papers. In spite of the wealth of structural data, the pathway of transmembrane movement for the large variety of structurally different ABCG2 substrates and the physiological lipid regulation of the transporter has not been elucidated. The complex molecular dynamics simulations presented here may provide a breakthrough in understanding the steps of the substrate transport process and its regulation by cholesterol. Our analysis revealed drug binding cavities other than the central binding site and delineated a putative dynamic transport pathway for substrates with variable structures. We found that membrane cholesterol accelerated drug transport by promoting the closure of cytoplasmic protein regions. Since ABCG2 is present in all major biological barriers and drug-metabolizing organs, influences the pharmacokinetics of numerous clinically applied drugs, and plays a key role in uric acid extrusion, this information may significantly promote a reliable prediction of clinically important substrate characteristics and drug-drug interactions.

Targeting the Acidic Tumor Microenvironment: Unexpected Pro-Neoplastic Effects of Oral NaHCO3 Therapy in Murine Breast Tissue

The acidic tumor microenvironment modifies malignant cell behavior. Here, we study consequences of the microenvironment in breast carcinomas. Beginning at carcinogen-based breast cancer induction, we supply either regular or NaHCO3-containing drinking water to female C57BL/6j mice. We evaluate urine and blood acid-base status, tumor metabolism (microdialysis sampling), and tumor pH (pH-sensitive microelectrodes) in vivo. Based on freshly isolated epithelial organoids from breast carcinomas and normal breast tissue, we assess protein expression (immunoblotting, mass spectrometry), intracellular pH (fluorescence microscopy), and cell proliferation (bromodeoxyuridine incorporation). Oral NaHCO3 therapy increases breast tumor pH in vivo from 6.68 ± 0.04 to 7.04 ± 0.09 and intracellular pH in breast epithelial organoids by ~0.15. Breast tumors develop with median latency of 85.5 ± 8.2 days in NaHCO3-treated mice vs. 82 ± 7.5 days in control mice. Oral NaHCO3 therapy does not affect tumor growth, histopathology or glycolytic metabolism. The capacity for cellular net acid extrusion is increased in NaHCO3-treated mice and correlates negatively with breast tumor latency. Oral NaHCO3 therapy elevates proliferative activity in organoids from breast carcinomas. Changes in protein expression patterns—observed by high-throughput proteomics analyses—between cancer and normal breast tissue and in response to oral NaHCO3 therapy reveal complex influences on metabolism, cytoskeleton, cell-cell and cell-matrix interaction, and cell signaling pathways. We conclude that oral NaHCO3 therapy neutralizes the microenvironment of breast carcinomas, elevates the cellular net acid extrusion capacity, and accelerates proliferation without net effect on breast cancer development or tumor growth. We demonstrate unexpected pro-neoplastic consequences of oral NaHCO3 therapy that in breast tissue cancel out previously reported anti-neoplastic effects.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells

Increased metabolic acid production and upregulation of net acid extrusion render pH homeostasis profoundly dysregulated in many cancers. Plasma membrane activity of vacuolar H+ ATPases (V-ATPases) has been implicated in acid extrusion and invasiveness of some cancers, yet often on the basis of unspecific inhibitors. Serving as a membrane anchor directing V-ATPase localization, the a subunit of the V0 domain of the V-ATPase (ATP6V0a1-4) is particularly interesting in this regard. Here, we map the regulation and roles of ATP6V0a3 in migration, invasion, and growth in pancreatic ductal adenocarcinoma (PDAC) cells. a3 mRNA and protein levels were upregulated in PDAC cell lines compared to non-cancer pancreatic epithelial cells. Under control conditions, a3 localization was mainly endo-/lysosomal, and its knockdown had no detectable effect on pHi regulation after acid loading. V-ATPase inhibition, but not a3 knockdown, increased HIF-1α expression and decreased proliferation and autophagic flux under both starved and non-starved conditions, and spheroid growth of PDAC cells was also unaffected by a3 knockdown. Strikingly, a3 knockdown increased migration and transwell invasion of Panc-1 and BxPC-3 PDAC cells, and increased gelatin degradation in BxPC-3 cells yet decreased it in Panc-1 cells. We conclude that in these PDAC cells, a3 is upregulated and negatively regulates migration and invasion, likely in part via effects on extracellular matrix degradation.

Deletion of the Na/HCO3 Transporter NBCn1 Protects Hippocampal Neurons from NMDA-induced Seizures and Neurotoxicity in Mice

The Na/HCO3 cotransporter NBCn1/SLC4A7 can affect glutamate neurotoxicity in primary cultures of rat hippocampal neurons. Here, we examined NMDA-induced neurotoxicity in NBCn1 knockout mice to determine whether a similar effect also occurs in the mouse brain. In primary cultures of hippocampal neurons from knockouts, NMDA had no neurotoxic effects, determined by lactate dehydrogenase release and nitric oxide synthase (NOS)-dependent cGMP production. Male knockouts and wildtypes (6–8 weeks old) were then injected with NMDA (75 mg/kg; ip) and hippocampal neuronal damages were assessed. Wildtypes developed severe tonic-clonic seizures, whereas knockouts had mild seizure activity (motionless). In knockouts, the NOS activity, caspase-3 expression/activity and the number of TUNEL-positive cells were significantly low. Immunochemical analysis revealed decreased expression levels of the NMDA receptor subunit GluN1 and the postsynaptic density protein PSD-95 in knockouts. Extracellular recording from hippocampal slices showed no Mg2+/NMDA-mediated epileptiform events in knockouts. In conclusion, these results show a decrease in NMDA neurotoxicity by NBCn1 deletion. Given that acid extrusion has been known to prevent pH decrease and protect neurons from acid-induced damage, our study presents novel evidence that acid extrusion by NBCn1 stimulates neurotoxicity.