Kathleen M. Randolph

Inhibitor Binding in the Human Renal Low- and High-Affinity Na+/Glucose Cotransporters

The kidney contains two Na+/glucose cotransporters, called SGLT2 and SGLT1, arranged in series along the length of the proximal tubule. The low-affinity transporter, SGLT2, is responsible for the reabsorption of most of the glucose in the kidney. There is recent interest in SGLT2 as a target for the treatment of type II diabetes using selective inhibitors based on the structure of the phenylglucoside, phlorizin (phloretin-2′-β-glucoside). In this study, we examined the inhibition of α-methyl-d-glucopyranose transport by phlorizin and a new candidate drug, sergliflozin-A [(2-[4-methoxyphenyl]methyl)phenyl β-d-glucopyranoside], in COS-7 cells expressing hSGLT1 and hSGLT2. Inhibition by phlorizin was competitive, with Ki values of 0.3 μM in hSGLT1 and 39 nM in hSGLT2. Inhibition by sergliflozin-A was also competitive, with Ki values of 1 μM in hSGLT1 and 20 nM in hSGLT2. Phloretin [3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone; the aglucone of phlorizin] was a less potent inhibitor, with IC50 values of 142 μM in hSGLT1 and 25 μM in hSGLT2. Site-directed mutagenesis of residues believed to be in the phlorizin binding site showed that only Cys610 is involved in inhibitor binding in the human transporters. Mutation of Cys610 in hSGLT1 to lysine resulted in an increased IC50 for all inhibitors. In contrast, mutagenesis of the analogous Cys615 in hSGLT2 produced the opposite effect, a decrease in IC50 for phlorizin and sergliflozin-A. The differences in the effects of the mutations between hSGLT1 and hSGLT2 suggest that this cysteine holds key residues in place rather than participating directly in inhibitor binding.

Muscle Protein Metabolism Responds Similarly to Exogenous Amino Acids in Healthy Younger and Older Adults during NO-Induced Hyperemia

The combination of increasing blood flow and amino acid (AA) availability provides an anabolic stimulus to the skeletal muscle of healthy young adults by optimizing both AA delivery and utilization. However, aging is associated with a blunted response to anabolic stimuli and may involve impairments in endothelial function. We investigated whether age-related differences exist in the muscle protein anabolic response to AAs between younger (30 ± 2 yr) and older (67 ± 2 yr) adults when macrovascular and microvascular leg blood flow were similarly increased with the nitric oxide (NO) donor, sodium nitroprusside (SNP). Regardless of age, SNP+AA induced similar increases above baseline (P ≤ 0.05) in macrovascular flow (4.3 vs. 4.4 ml·min(-1)·100 ml leg(-1) measured using indocyanine green dye dilution), microvascular flow (1.4 vs. 0.8 video intensity/s measured using contrast-enhanced ultrasound), phenylalanine net balance (59 vs. 68 nmol·min(-1)·100 ml·leg(-1)), fractional synthetic rate (0.02 vs. 0.02%/h), and model-derived muscle protein synthesis (62 vs. 49 nmol·min(-1)·100 ml·leg(-1)) in both younger vs. older individuals, respectively. Provision of AAs during NO-induced local skeletal muscle hyperemia stimulates skeletal muscle protein metabolism in older adults to a similar extent as in younger adults. Our results suggest that the aging vasculature is responsive to exogenous NO and that there is no age-related difference per se in AA-induced anabolism under such hyperemic conditions.

Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

The Na+/dicarboxylate co-transporter, NaDC-1, from the kidney and small intestine, transports three sodium ions together with one divalent anion substrate, such as succinate2–. A previous study (Pajor, A. M. (2001) J. Biol. Chem. 276, 29961–29968), identified four amino acids, Ser-478, Ala-480, Ala-481, and Thr-482, near the extracellular end of transmembrane helix (TM) 9 that are likely to form part of the permeation pathway of the transporter. All four cysteine-substituted mutants were sensitive to inhibition by the membrane-impermeant reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) and protected by substrate. In the present study, we continued the cysteine scan through extracellular loop 5 and TM10, from Thr-483 to Val-528. Most cysteine substitutions were well tolerated, although cysteine mutations of some residues, particularly within the TM, produced proteins that were not expressed on the plasma membrane. Six residues in the extracellular loop (Thr-483, Thr-484, Leu-485, Leu-487, Ile-489, and Met-493) were sensitive to chemical labeling by MTSET, depending on the conformational state of the protein. Transport inhibition by MTSET could be prevented by substrate regardless of temperature, suggesting that the likely mechanism of substrate protection is steric hindrance rather than large-scale conformational changes associated with translocation. We conclude that extracellular loop 5 in NaDC-1 appears to have a functional role, and it is likely to be located in or near the substrate translocation pore in the protein. Conformational changes in the protein affect the accessibility of the residues in extracellular loop 5 and provide further evidence of large-scale changes in the structure of NaDC-1 during the transport cycle.

Inhibition of the Na+/Dicarboxylate Cotransporter by Anthranilic Acid Derivatives

The Na+/dicarboxylate cotransporter NaDC1 absorbs citric acid cycle intermediates from the lumen of the small intestine and kidney proximal tubule. No effective inhibitor has been identified yet, although previous studies showed that the nonsteroidal anti-inflammatory drug, flufenamate, inhibits the human (h) NaDC1 with an IC50 value of 2 mM. In the present study, we have tested compounds related in structure to flufenamate, all anthranilic acid derivatives, as potential inhibitors of hNaDC1. We found that N-(p-amylcinnamoyl) anthranilic acid (ACA) and 2-(p-amylcinnamoyl) amino-4-chloro benzoic acid (ONO-RS-082) are the most potent inhibitors with IC50 values lower than 15 μM, followed by N-(9-fluorenylmethoxycarbonyl)-anthranilic acid (Fmoc-anthranilic acid) with an IC50 value of ∼80 μM. The effects of ACA on NaDC1 are not mediated through a change in transporter protein abundance on the plasma membrane and seem to be independent of its effect on phospholipase A2 activity. ACA acts as a slow inhibitor of NaDC1, with slow onset and slow reversibility. Both uptake activity and efflux are inhibited by ACA. Other Na+/dicarboxylate transporters from the SLC13 family, including hNaDC3 and rbNaDC1, were also inhibited by ACA, ONO-RS-082, and Fmoc-anthranilic acid, whereas the Na+/citrate transporter (hNaCT) is much less sensitive to these compounds. The endogenous sodium-dependent succinate transport in Caco-2 cells is also inhibited by ACA. In conclusion, ACA and ONO-RS-082 represent promising lead compounds for the development of specific inhibitors of the Na+/dicarboxylate cotransporters.

Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.

Efficacy of Testosterone plus NASA Exercise Countermeasures during Head-Down Bed Rest

Introduction Prolonged confinement to head-down bed rest (HDBR) results in musculoskeletal losses similar to those observed during long-duration space flight. Exercise countermeasures by themselves have not completely prevented the deleterious losses in muscle mass or function in HDBR or space flight. Purpose The objective was to investigate the safety and efficacy of intermittent, low-dose testosterone treatment in conjunction with NASA exercise (SPRINT) countermeasures during 70 d of 6° HDBR. Methods Healthy men (35 ± 8 yr) were randomized into one of three groups that remained inactive (CON) or performed exercise 6 d·wk−1 in addition to receiving either placebo (PEX) or testosterone treatment (TEX, 100 mg·wk−1). Testosterone/placebo injections were administered once a week for 2 wk, followed by 2 wk off and so on, during HDBR. Results Total, leg, and trunk lean body mass (LBM) consistently decreased in CON, increased in TEX, and had little or no changes in PEX. Total, leg, and trunk fat mass consistently increased in CON and PEX and decreased in TEX. Leg strength decreased in CON, whereas PEX and TEX were protected against loss in strength. Changes in leg LBM correlated positively with changes in leg muscle strength. Conclusions Addition of a testosterone countermeasure enhanced the preventative actions of exercise against body composition changes during long-term HDBR in healthy eugonadal men. This is the first report to demonstrate that cycled, low-dose testosterone treatment increases LBM under conditions of strict exercise control. These results are clinically relevant to the development of safe and effective therapies against muscle atrophy during long-term bed rest, aging, and disease where loss of muscle mass and strength is a risk. The potential space flight applications of such countermeasure combinations deserve further investigations.

A randomized trial of adjunct testosterone for cancer-related muscle loss in men and women: Testosterone therapy ameliorates cancer-related muscle loss

Cancer cachexia negatively impacts cancer‐related treatment options, quality of life, morbidity, and mortality, yet no established therapies exist. We investigated the anabolic properties of testosterone to limit the loss of body mass in late stage cancer patients undergoing standard of care cancer treatment.