Absence of Na+/sugar cotransport activity in Barrett’s metaplasia

Abstract

AIM: To evaluate the presence of Na⁺-dependent, active, sugar transport in Barrett's epithelia as an intestinal biomarker, based on the well-documented, morphological intestinal phenotype of Barrett's esophagus (BE).

METHODS: We examined uptake of the nonmeta-bolizable glucose analogue, alpha-methyl-D-glucoside (AMG), a substrate for the entire sodium glucose cotransporter (SGLT) family of transport proteins. During upper endoscopy, patients with BE or with uncomplicated gastroesophageal reflux disease (GERD) allowed for duodenal, gastric fundic, and esophageal mucosal biopsies to be taken. Biopsies were incubated in bicarbonate-buffered saline (KRB) containing 0.1 mmol/L ¹⁴C-AMG for 60 min at 20°C. Characterized by abundant SGLT, duodenum served as a positive control while gastric fundus and normal esophagus, known to lack SGLT, served as negative controls.

RESULTS: Duodenal biopsies accumulated 249.84 ± 35.49 (SEM) picomoles AMG/&mgr;g DNA (n = 12), gastric fundus biopsies 36.20 ± 6.62 (n = 12), normal esophagus 12.10 ± 0.59 (n = 3) and Barrett’s metaplasia 29.79 ± 5.77 (n = 8). There was a statistical difference (P < 0.01) between biopsies from duodenum and each other biopsy site but there was no statistically significant difference between normal esophagus and BE biopsies. 0.5 mmol/L phlorizin (PZ) inhibited AMG uptake into duodenal mucosa by over 89%, but had no significant effect on AMG uptake into gastric fundus, normal esophagus, or Barrett’s tissue. In the absence of Na⁺ (all Na⁺ salts replaced by Li⁺ salts), AMG uptake in duodenum was decreased by over 90%, while uptake into gastric, esophageal or Barrett’s tissue was statistically unaffected.

CONCLUSION: Despite the intestinal enterocyte phenotype of BE, Na⁺-dependent, sugar transport activity is not present in these cells.

Key Words: Barrett’s esophagus, Cancer, Biomarker, Sodium glucose cotransporters, Glucose transport, Alpha-methyl-D-glucoside, Phlorizin, Esophagus, Metaplasia

INTRODUCTION

Esophageal adenocarcinoma, which has increased 4-fold between 1973 and 2002, is escalating at an incidence rate which exceeds that of any other malignancy[1–3]. Generally detected at a late stage, esophageal adenocarcinoma is extremely lethal, with a 5-year survival rate of just 10%-20%[4]. Barrett’s esophagus (BE), a condition charac-terized by a columnar, intestinal-like metaplasia of the normally stratified squamous distal esophagus, confers a dramatically increased risk of progression to esophageal adenocarcinoma and serves currently as the most important clinical risk factor for development of such malignancy[2]. In fact, the risk of esophageal adenocarcinoma in BE patients is 30-120 times that of the general population[25].

BE may be present in 3% of the western hemisphere population and in at least 10% of gastroesophageal reflux disease (GERD) patients[1]. It is impractical to attempt to identify the BE population by means of endoscopic surveillance of all GERD patients, considering that there are 30 million Americans with GERD[6]. Only 5% of patients presenting with esophageal adenocarcinoma are preceded by a BE diagnosis made by upper endoscopy, further illustrating the logistical difficulties in screening a large population by this means[4]. Thus, it will be valuable to find and establish biomarkers which may be used to identify BE, simply, safely, and inexpensively, in order to appropriately avert esophageal adenocarcinoma.

Current cellular biomarkers of BE include sucrase-isomaltase (SI) and dipeptidyl peptidase IV (DPP), enzymes which are present in about 60% of BE patients[7]. Cdx2, which encodes an intestine-specific transcription factor, is another possible BE biomarker presently under investigation[8]. Villin, a cytoskeletal protein related to microvilli, and Ep-CAM, a glandular epithelial glycoprotein are also possible BE biomarkers[9]. Other potential markers of BE are the deletion of p53 tumor suppressor gene, deletion or hypermethylation of p16, changes in COX-2 expression, and increases in cell proliferation and cell cycling markers like cyclin D1[24]. Two inherent disadvantages of these markers are: (1) each individually will not be found in every Barrett’s biopsy and (2) all will require tissue biopsies to be taken (upper endoscopies to be performed).

The heterogeneity of BE is evidenced by the presence of columnar non-goblet cells intermixed with the intestinal-like goblet cells in the metastatic Barrett’s population[7]. The specialized epithelial columnar cells are known to express the small intestinal enzymes, SI and dipeptidyl peptidase, and perhaps represent an “incomplete” stage of intestinal metaplasia[7]. Additionally, heterogeneity exists with regard to villin and Ep-CAM expression in Barrett’s cells, indicating variations in differentiation state within the Barrett’s epithelium[9].

BE, which has a patchy, irregular appearance, typically consists of three distinct cell morphologies: an atrophic gastric fundus type, a junctional gastric cardia type, and an intestinal-like population containing goblet cells and also non-goblet columnar cells (both mucus-containing and pseudo-absorptive in nature)[56910]. Of these three, only the goblet cell population exhibiting an intestinal phenotype is currently definitively associated with increased risk of malignancy and is thus exclusively required for clinical BE diagnosis[68]. The histological detection of mucin, which can be achieved in dramatic fashion by Alcian blue staining defines the ‘goblet cell metaplasia’ character of true BE[11].

Of course, histology requires mucosal biopsies, which can be obtained only through upper endoscopy. A major emphasis of current research in BE is to define new biomarkers which may lead to screening techniques not dependent upon endoscopy and biopsies, due to the expense of anesthesia and risk of the procedure.

The intestinal-like phenotype of BE prompted our group to test for the presence of Na⁺-dependent, active, sugar transport activity (the SGLT family of proteins) in BE as a potential biomarker. Glucose is absorbed in the intestine by SGLT through an energy-, concentration- and Na⁺-dependent fashion[12–14]. SGLT is specific to the differentiated, absorptive epithelial cells in the small intestine, kidney proximal tubule, and a few other epithelial cell types and is absent in the stomach, normal esophagus, and most other somatic cells[15–18]. The nonmetabolizable glucose analogue, alpha-methyl-D-glucoside (AMG), is a substrate specific for the sodium-dependent (active) glucose cotransporter (SGLT) family of transport proteins, since a free hydroxyl on carbon 1 is not required for uptake by SGLT[19]. AMG is, however, a very poor substrate for the more ubiquitous facilitated diffusion transporters of the GLUT family[20]. Phlorizin (PZ) is a competitive inhibitor of intestinal active glucose transport but is not transported itself by the Na⁺/glucose cotransporter[21].

MATERIALS AND METHODS

Biopsy acquisition

Under an IRB-approved protocol, patients with and without BE, presenting to the Lankenau Hospital Gastroenterology group for upper endoscopy for screening or diagnostic purposes relating to reflux, gastritis, or dysphagia, provided written informed consent for additional biopsies to be taken during upper endoscopy. The patient population of 23 people included 9 females and 14 males, ranging in age from 47 to 81 years. Ten of the study patients were known to have BE, as confirmed by prior positive Alcian blue staining of endoscopic biopsies.

For simple AMG uptake studies, a total of nine biopsies were taken, three each from duodenum, gastric fundus, and Barrett’s metaplasia or normal esophagus. For PZ inhibition and Li⁺ saline/Na⁺ saline studies, a total of eight biopsies were taken from each patient, four each from two of the above listed sites.Duodenum, known to have abundant SGLT, served as a positive control. Gastric fundus and normal esophagus served as negative controls. These tissues are known not to exhibit Na⁺/glucose cotransport activity. By evaluating each BE biopsy individually for uptake of ¹⁴C-AMG, we address the possibility of AMG uptake being expressed in only certain regions of Barrett’s epithelium in any individual Barrett’s patient.

¹⁴C-AMG uptake studies

For AMG uptake studies, endoscopic biopsies were placed immediately in ice cold, freshly oxygenated bicarbonate-buffered saline (KRB) and transported from the endoscopy procedure room to the laboratory. Because glucose would interfere with AMG uptake, 5 mmol/L sodium acetate was used to replace glucose as a metabolic substrate in the saline. KRB contained 112 mmol/L NaCl, 5 mmol/L KCl, 1.25 mmol/L CaCl₂∙2H₂O, 1.1 mmol/L MgCl₂∙6H₂O, 2.4 mmol/L Na₂HPO₄, 0.6 mmol/L NaH₂PO₄∙H₂O, 25 mmol/L NaHCO_3, 5 mmol/L sodium acetate.

Biopsies were incubated in KRB containing 0.1 mmol/L ¹⁴C-AMG at 20°C for 60 min while being shaken and aerated with 5% CO₂/95% O₂. Methyl-alpha-D [U-¹⁴C] glucopyranoside (AMG) was purchased from Amersham Biosciences (UK). Biopsies were copiously rinsed with 4°C saline (0.154 mol/L NaCl) at the end of the incubation to remove extracellular ¹⁴C-AMG, then a wet weight was recorded followed by addition of biopsy to 0.4 mmol/L perchloric acid for release of intracellular radioactivity, and precipitation (and later measurement) of total DNA by the diphenylamine reaction[22]. Intracellular ¹⁴C-AMG released from the samples was quantified by scintillation counting and then normalized per wet weight and per total amount of DNA.

PZ inhibition studies

To confirm the absence of AMG uptake by SGLT, a series of experiments were performed using PZ, a specific, competitive inhibitor of SGLT. PZ dihydrate was purchased from Sigma-Aldrich (St. Louis, MO). For PZ inhibition studies, biopsies were collected in a similar fashion but were pre-incubated in KRB with or without 0.5 mmol/L PZ (and without AMG) for 15 min, while being shaken and aerated with 5% CO₂/95% O₂. After 15 min, 0.1 mmol/L ¹⁴C-AMG ± 0.5 mmol/L PZ was added to the saline and samples were incubated for an additional 60 min. After the incubation period, samples were similarly rinsed, weighed, and digested for determination of ¹⁴C-AMG uptake per sample wet weight.

Sodium dependence studies

To further confirm a lack of Na⁺/sugar cotransport activity, a series of experiments were performed in which lithium salts replaced sodium salts in the incubation medium. In addition, KRB contained 5 mmol/L lithium acetate in place of sodium acetate. Biopsies were similarly incubated with ¹⁴C-AMG (in KRB with either sodium or lithium salts), rinsed, and weighed for determination of ¹⁴C-AMG uptake per sample wet weight.

RESULTS

Na⁺/sugar cotransport was assayed by measuring uptake of the nonmetabolizable glucose analogue, AMG, a substrate for all three known SGLTs, with K_m values of 0.4 mmol/L for SGLT1 and 2 mmol/L for SGLT2 and SGLT3[23]. AMG is not a substrate for facilitated diffusional transporters (GLUT1, etc) and thus cannot easily efflux from the cell[20], meaning that AMG will accumulate to high intracellular concentrations in cells expressing SGLT. AMG uptake by duodenal biopsy samples was 17-fold greater than gastric biopsies, indicative of the known presence of SGLT in duodenal tissue (Table 1). Dramatically lower AMG uptake in gastric and normal esophageal biopsies correlated with the known absence of SGLT in these tissues. Barrett’s biopsies were unable to concentrate AMG to the levels exhibited by duodenal biopsies, suggesting lack of Na⁺-dependent sugar uptake in Barrett’s tissue.

Table 1 Uptake of AMG by duodenal, gastric fundic, and esophageal mucosal biopsies (mean ± SE).

	pmoles AMG mg wet wt	pmoles AMG/&mgr;g DNA
Duodenum	711.53 ± 121.63 (11)	249.84 ± 35.49 (12)
Fundus	41.04 ± 2.35 (12)b	36.20 ± 6.62 (12)b
Esophagus	45.52 ± 3.76 (3)a	12.10 ± 0.59 (3)d
Barrett’s	50.27 ± 4.32 (9)b	29.79 ± 5.77 (8)b

The number in parenthesis represents the biopsies tested. Three biopsies were taken from each tissue site of each patient.

^bP < 0.001 vs duodenum;

^aP < 0.02 vs duodenum;

^dP < 0.01 vs duodenum (Student's t-test). There was no significant difference (P > 0.05) between Barrett’s esophagus biopsies and biopsies of fundus or normal esophagus.

To confirm this absence of AMG uptake in Barrett’s epithelium, an additional series of experiments was performed using PZ, a competitive inhibitor of sugar uptake specifically by SGLT[24–27]. AMG uptake by duodenal biopsies was inhibited over 85% by 0.5 mmol/L PZ, consistent with the known presence of Na⁺/sugar cotransport in duodenum (Table 2). AMG uptake in gastric and normal esophageal biopsies was not significantly inhibited by PZ. This same lack of PZ inhibition of AMG uptake into biopsies of Barrett’s metaplasia is a further indication of the lack of active sugar transport activity in Barrett’s epithelium.

Table 2 Inhibition of AMG uptake by 0.5 mmol/L phlorizin in duodenal, gastric fundic, and esophageal mucosal biopsies (mean ± SE).

	Control	Phlorizin
Duodenum	593.34 ± 120.23 (7)	65.05 ± 14.41 (7)b
Fundus	35.00 ± 1.82 (8)	30.27 ± 4.23 (8)a
Esophagus	36.73 ± 4.85 (8)	27.20 ± 2.63 (8)a
Barrett’s	53.09 ± 5.57 (6)	42.66 ± 4.37 (7)a

The number in parenthesis represents the biopsies tested.

^bP < 0.001 vs matched control (Student's t-test);

^aP > 0.05 vs control.

As final verification of the lack of Na⁺/sugar cotransport in Barrett’s epithelium, additional experiments were performed in which lithium salts replaced sodium salts in the incubation saline. Intracellular uptake of AMG into duodenal biopsies in Na⁺-free (Li⁺) saline was only 10% of the levels in Na⁺ saline (Table 3). However, the presence of Na⁺ did not significantly affect AMG uptake into gastric, esophageal, or Barrett’s tissue biopsies. This lack of Na⁺-dependence of sugar transport further confirms the lack of active sugar transport in Barrett’s epithelium.

Table 3 Sodium dependence or independence of AMG uptake by duodenal, gastric fundic, and esophageal mucosal biopsies (mean ± SE).

	Lithium	Sodium
Duodenum	49.31 ± 5.86 (10)b	544.85 ± 110.20 (10)d
Fundus	35.20 ± 2.73 (8)	41.15 ± 1.91 (7)
Esophagus	44.83 ± 4.03 (9)	41.56 ± 5.07 (9)
Barrett’s	40.47 ± 1.47 (5)	48.03 ± 11.72 (5)

The number in parentheses represents the biopsies tested.

^bP < 0.001,

^dP < 0.001 vs all other samples. There was no significant difference between lithium and sodium samples.

DISCUSSION

The inability of Barrett’s tissue to concentrate AMG, the lack of inhibition by PZ, and the absence of Na⁺-dependence all indicate the absence of an active sugar transport system in BE. Despite the intestinal phenotype ascribed to Barrett’s metaplasia, with classic intestinal markers like SI activity and mucin secretion, Barrett’s biopsies appear to lack activity associated with SGLT, the active sugar transporter that typifies normal small intestine. The absence of SGLT activity in Barrett’s tissue highlights the fact that Barrett’s cells are not simply an intestinal cell type in the distal esophagus, even though Barrett’s tissue has other intestinal-like characteristics.

Our results, however, do leave open the possibility, though unlikely, that SGLT is present in the plasma membrane or cells of Barrett’s epithelium though it is not functional. Thus, the assays described in this paper were strictly limited to determination of the functional presence of SGLT. Using duodenal biopsies, our group obtained only inconclusive and disappointing results with two commercial SGLT antibodies, therefore not allowing a parallel immunological approach to this question.

Barrett’s epithelium develops as a result of damage to the stratified squamous tissue of the distal esophagus by acid and bile from reflux[5]. Because of the unique, columnar, intestinal-like phenotype of Barrett’s epithelium, it is widely accepted that Barrett’s epithelium arises from clonal growth of pluripotent stem cells rather than growth from adjacent inflamed esophageal squamous epithelia, as the “de novo” metaplasia theory suggests[69]. The duct cell metaplasia theory posits that Barrett’s cells derive from stem cells in esophageal glands that colonize the esophagus when the mucosa is damaged[46]. Finally, the transitional zone metaplasia theory states that, when exposed to bile and acid, the cells of the distal esophagus are replaced by stem cells from the gastroesophageal junction[56]. Whatever the origin, BE is a “successful adaptation” for protection of the esophagus from further reflux damage, saving the individual with reflux from a future of ulceration and strictures, but carrying with it an increased cancer risk[5].

SGLT is expressed on the apical plasma membrane of absorptive epithelial cells in the small intestine but is not expressed by goblet cells[171828]. Also, different rates of Na⁺-dependent glucose transport have been observed in mature cells from the villi of intestine and in immature crypt cells, suggesting that differentiation and enterocyte development state may contribute to SGLT expression and affinity levels[29–31]. One must therefore consider that not all small intestinal cell types possess Na⁺/glucose cotransport and that BE may be reflective of these cell types.

Although SGLT may not serve as a biomarker for BE, our group and others have collected evidence that the transporter may be present in intestinalized gastric mucosa and may potentially indicate metaplastic changes related to gastric adenocarcinoma[32]. As aforementioned, SGLT is normally not present in the stomach. However, biopsies from a patient with a large, grossly-visible, hyperplastic gastric polyp, exhibited ¹⁴C-AMG uptake that was inhibited by PZ and approximately five times greater than uptake by the patient’s normal esophageal biopsies (Data not shown).

In summary, despite the intestinal phenotype ascribed to Barrett’s metaplasia with classic intestinal markers like SI activity and mucin secretion, Barrett’s biopsies appear to lack activity associated with SGLT, the active sugar transporter present in normal small intestine, and thus cannot serve as a BE biomarker. The absence of SGLT activity in Barrett’s biopsies highlights the fact that, although it has other intestinal-like characteristics, Barrett’s cells are not simply an intestinal cell type in the distal esophagus, but should rather be treated as a unique entity in future considerations.

COMMENTS

Background

In order to effectively avert esophageal adenocarcinoma, it is necessary to find and establish biomarkers to identify Barrett’s esophagus (BE). The purpose of this study was to evaluate the presence of Na⁺-dependent, active, sugar transport in Barrett’s epithelia as an intestinal biomarker, based on the well-documented, morphological intestinal phenotype of BE.

Research frontiers

To our knowledge, this is the first paper to investigate Na⁺-dependent, active, sugar transport in Barrett’s epithelia as an intestinal biomarker.

Innovations and breakthroughs

Despite the intestinal phenotype ascribed to Barrett’s metaplasia with classic intestinal markers like sucrase-isomaltase (SI) activity and mucin secretion, Barrett’s biopsies appear to lack activity associated with SGLT, the active sugar transporter present in normal small intestine, and thus cannot serve as a BE biomarker. The absence of SGLT activity in Barrett’s biopsies highlights the fact that, although it has other intestinal-like characteristics, Barrett’s cells are not simply an intestinal cell type in the distal esophagus, but should rather be treated as a unique entity in future considerations.

Applications

While our studies show a lack of functional SGLT1 protein in BE, it may also be valuable to determine the expression of SGLT1 mRNA in Barrett’s tissue by RT-PCR.

Peer review

This nice study set out to assess by functional criteria whether SGLT1 is expressed in Barrett's metaplasia. It well evaluated the presence of Na⁺-dependent, active, sugar transport in Barrett’s epithelia as an intestinal biomarker.