Diabulimia: A Risky Trend Among Adults with Type 1 Diabetes Mellitus.

OBJECTIVE: Poor adherence leads to worse glycemic control and increased complications in patients with type 1 diabetes mellitus (T1DM). Diabulimia characterizes patients with T1DM who skip or use less insulin for weight loss purposes. The study objectives were to determine: (1) the prevalence of diabulimia among adult patients with T1DM, (2) compare patients with and without diabulimia, and (3) identify factors that may place individuals at higher risk of diabulimia. METHODS: A 40-item, web-based survey was administered to 21 T1DM discussion boards, Listservs, and social media outlets. The survey assessed demographics, diabetes management, psychiatric diagnoses, and screened for diabulimia. Individuals who reported intentionally skipping or using less insulin than directed for the purpose of weight loss or to prevent weight gain in the past 12 months were classified as having diabulimia. RESULTS: Of the 225 participants who completed the survey, 8.9% had diabulimia. Patients with diabulimia had elevated hemoglobin A1C (A1C) levels (8.4% vs 6.9%; P = .014), higher rates of a diabetes-related emergency department visits or hospitalization (30.0% vs 13.2%; P = .042), and higher rates of a major depressive disorder diagnosis (40.0% vs 11.5%; P < .001) than patients without diabulimia. Factors associated with diabulimia included high A1C levels (odds ratio, 1.43; 95% CI [1.08-1.91]; P = .014) and a major depressive disorder diagnosis (odds ratio, 4.87; 95% CI [1.31-18.22]; P = .018). CONCLUSION: Approximately 1 in 11 adult patients with T1DM screened positive for diabulimia. Higher A1C levels and a diagnosis of major depressive disorder were associated with diabulimia.

A proton-coupled folate transporter mutation causing hereditary folate malabsorption locks the protein in an inward-open conformation.

The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.

Concentrative Transport of Antifolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer.

The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folate-specific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-buffered medium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na+/H+ exchanger(s); it was eliminated by inhibitors of Na+/H+ exchanger (s) or Na+/K+ ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.

Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation.

The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A1) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These posttranslational modifications limit the accessibility of the cysteine residues to sulfhydryl-reactive reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a posttranslationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a posttranslational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.

Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking.

The proton-coupled folate transporter (PCFT, SLC46A1) is required for intestinal folate absorption and folate homeostasis in humans. A homology model of PCFT, based upon theEscherichia coliglycerol 3-phosphate transporter structure, predicted that PCFT transmembrane domains (TMDs) 1, 2, 7, and 11 form an extracellular gate in the inward-open conformation. To assess this model, five residues (Gln(45)-TMD1, Asn(90)-TMD2, Leu(290)-TMD7, Ser(407)-TMD11 and Asn(411)-TMD11) in the predicted gate were substituted with Cys to generate single and nine double mutants. Transport function of the mutants was assayed in transient transfectants by measurement of [(3)H]substrate influx as was accessibility of the Cys residues to biotinylation. Pairs of Cys residues were assessed for spontaneous formation of a disulfide bond, induction of a disulfide bond by oxidization with dichloro(1,10-phenanthroline)copper (II) (CuPh), or the formation of a Cd(2+)complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/S407C pair and a CuPh- and Cd(2+)-induced disulfide bond and complex, respectively, for the Q45C/L290C and L290C/N411C pairs. The decrease in activity induced by cross-linkage of the Cys residue pairs was due to a decrease in the influxVmaxconsistent with restriction in the mobility of the transporter. The presence of folate substrate decreased the CuPh-induced inhibition of transport. Hence, the data support the glycerol 3-phosphate transporter-based homology model of PCFT and the presence of an extracellular gate formed by TMDs 1, 2, 7, and 11.

Role of the tryptophan residues in proton-coupled folate transporter (PCFT-SLC46A1) function.

The proton-coupled folate transporter (PCFT) mediates folate absorption across the brush-border membrane of the proximal small intestine and is required for folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues were assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCFT mutant located in the 4th external loop between the 7th and 8th transmembrane helices. Despite its aqueous location, function could only be fully preserved with Phe and, to a lesser extent, Ala substitutions. There was a 6.5-fold decrease in the pemetrexed influx Vmax and a 3.5- and 6-fold decrease in the influx Kt and Ki, respectively, for the W299S PCFT. The data indicate that the hydrophobicity of the W299 residue is important for function suggesting that during the transport cycle this residue interacts with the lipid membrane thereby impacting on the oscillation of the carrier and, indirectly, on the folate binding pocket.

Identification of Tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1).

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/Ki for [(3)H]methotrexate and [(3)H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [(3)H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carrier's mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.