Alternative Splicing and Cleavage of GLUT8.

The GLUT (SLC2) family of membrane-associated transporters are described as glucose transporters. However, this family is divided into three classes and, though the regulated transporter activity of class I proteins is becoming better understood, class III protein functions continue to be obscure. We have cataloged the relative expression and splicing of SLC2 mRNA isomers in tumors and normal tissues, with a focus on breast tumors and cell lines. mRNA for the class III protein GLUT8 is the predominant SLC2 species expressed alongside GLUT1 in many tissues, but GLUT8 mRNA exists mostly as an untranslated splice form in tumors. We confirm that GLUT8 is not presented at the cell surface and does not transport glucose directly. However, we reveal a lysosome-dependent reaction that cleaves the GLUT8 protein and releases the carboxy-terminal peptide to a separate vesicle population. Given the localization of GLUT8 at a major metabolic hub (the late endosomal/lysosomal interface) and its regulated cleavage reaction, we evaluated TXNIP-mediated hexosamine homeostasis and speculate that GLUT8 may function as a sensory component of this reaction.

Human adipose beiging in response to cold and mirabegron.

BACKGROUND: The induction of beige adipocytes in s.c. white adipose tissue (WAT) depots of humans is postulated to improve glucose and lipid metabolism in obesity. The ability of obese, insulin-resistant humans to induce beige adipose tissue is unknown. METHODS: We exposed lean and obese research participants to cold (30-minute ice pack application each day for 10 days of the upper thigh) or treated them with the ß3 agonist mirabegron. We determined beige adipose marker expression by IHC and quantitative PCR, and we analyzed mitochondrial bioenergetics and UCP activity with an Oxytherm system. RESULTS: Cold significantly induced UCP1 and TMEM26 protein in both lean and obese subjects, and this response was not associated with age. Interestingly, these proteins increased to the same extent in s.c. WAT of the noniced contralateral leg, indicating a crossover effect. We further analyzed the bioenergetics of purified mitochondria from the abdominal s.c. WAT of cold-treated subjects and determined that repeat ice application significantly increased uncoupled respiration, consistent with the UCP1 protein induction and subsequent activation. Cold also increased State 3 and maximal respiration, and this effect on mitochondrial bioenergetics was stronger in summer than winter. Chronic treatment (10 weeks; 50 mg/day) with the ß3 receptor agonist mirabegron induces UCP1, TMEM26, CIDEA, and phosphorylation of HSL on serine660 in obese subjects. CONCLUSION: Cold or ß3 agonists cause the induction of beige adipose tissue in human s.c. WAT; this phenomenon may be exploited to increase beige adipose in older, insulin-resistant, obese individuals. TRIAL REGISTRATION: Clinicaltrials.gov NCT02596776, NCT02919176. FUNDING: NIH (DK107646, DK112282, P20GM103527, and by CTSA grant UL1TR001998).

Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases.

BACKGROUND: Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan normally expressed primarily by epithelia and plasma cells, is aberrantly induced in stromal fibroblasts of breast carcinomas. Stromal fibroblast-derived Sdc1 participates in paracrine growth stimulation of breast carcinoma cells and orchestrates stromal extracellular matrix fiber alignment, thereby creating a migration and invasion-permissive microenvironment. Here, we specifically tested the role of stromal Sdc1 in metastasis. METHODS: The metastatic potential of the aggressive mouse mammary carcinoma cell lines, 4T1 and E0776, was tested in wild-type and genetically Sdc1-deficient host animals. Metastatic lesions were characterized by immunohistochemical analysis. RESULTS: After orthotopic inoculation, the lung metastatic burden was reduced in Sdc1-/- animals by 97% and more than 99%, in BALB/cJ and C57BL/6 animals, respectively. The difference in metastatic efficiency was maintained when the tumor cells were injected into the tail vein, suggesting that host Sdc1 exerts its effect during later stages of the metastatic cascade. Co-localization studies identified Sdc1 expression in stromal fibroblasts within the metastatic microenvironment and in normal airway epithelial cells but not in other cells (endothelial cells, α-smooth muscle actin positive cells, leucocytes, macrophages). The Ki67 proliferation index and the rate of apoptosis of the metastatic tumor cells were diminished in Sdc1-/- vs. Sdc1+/+ animals, and leucocyte density was indistinguishable. Sdc1-mediated metastatic efficiency was abolished when the animals were housed at a thermoneutral ambient temperature of 31 °C, suggesting that the host Sdc1 effect on metastasis requires mild cold stress. CONCLUSIONS: In summary, Sdc1 is induced in the lung microenvironment after mammary carcinoma cell dissemination and promotes outgrowth of metastases in a temperature-dependent manner.

Expression levels of the ABCG2 multidrug transporter in human erythrocytes correspond to pharmacologically relevant genetic variations.

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

PI3-kinase and mTOR inhibitors differently modulate the function of the ABCG2 multidrug transporter.

The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.

A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome.

Scott syndrome (SS) is a bleeding disorder characterized by a failure to expose phosphatidylserine (PS) to the outer leaflet of the platelet plasma membrane. Because the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) is implicated in the exofacial translocation of PS, we assessed its role in the pathophysiology of a patient with SS. Substantially reduced levels of ABCA1 mRNA were found in the patient's leukocytes, compared with controls. The SS patient was heterozygous for a novel missense mutation c.6064G>A (ABCA1 R1925Q), absent from unaffected family members and controls. Both mutant and wild-type alleles were reduced in mRNA expression, and no causative mutation for this phenomenon was identified in the ABCA1 gene or its proximal promoter, suggesting a putative second mutation in a trans-acting regulatory gene may also be involved in the disorder in this patient. In vitro expression studies showed impaired trafficking of ABCA1 R1925Q to the plasma membrane. Overexpression of wild-type ABCA1 in SS lymphocytes complemented the Ca2+-dependent PS exposure at the cell surface. These data identify a mutation in ABCA1 that contributes to the defective PS translocation phenotype in our patient with SS.