Báo cáo khoa học: Investigation of the effects of sulfonylurea exposure on pancreatic beta cell metabolism pptx

Experiments investigating the effects of chronic exposure of pancre-atic islets and beta cell lines to sulfonylureas in vitro have shown that the desensitization in certain cases is not

pancreatic beta cell metabolism

Lorraine Brennan1, Chandralal Hewage1, J P G Malthouse1, Neville H McClenaghan2,

Peter R Flatt2and Philip Newsholme1

1 UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Ireland

2 School of Biomedical Sciences, University of Ulster, Coleraine, UK

Sulfonylureas are a major class of potent

insulinotrop-ic drugs that are used extensively in the treatment of

patients with type 2 diabetes These drugs stimulate

secretion of insulin from the pancreatic beta cell,

primarily by interacting with the high-affinity

sulfonyl-urea receptor (SUR1) subunit of the beta cell

ATP-sensitive potassium ion channel (KATP) channel

Sulfonylurea drugs differ in their specificity for the

SUR1 subunit The interaction with the KATP channel

closes the channel, causing membrane depolarization

and subsequent opening of voltage-dependent Ca2+

channels [1] The resulting influx of Ca2+ leads to a

rapid rise in intracellular Ca2+ concentration and

trig-gers insulin secretion [2,3] However, a growing body

of evidence suggests that the sulfonylureas additionally

act in a KATPchannel-independent manner by directly

interacting with the secretory machinery, and it has

been suggested that this effect is indirectly dependent

on protein kinase C activation [4–6] Interestingly, a recent study has proposed a mechanism by which sulfonylureas could be transported across the plasma membrane and ultimately interact with intracellular sites regulating insulin exocytosis [7]

Treatment of patients with sulfonylureas for pro-longed periods (several years) often results in impaired sulfonylurea-induced insulin secretion [8,9] Although the exact reasons underlying this phenomenon remain unclear, it is now believed that this may be at least partly attributed to desensitization of the pancreatic beta cells to the actions of these drugs Experiments investigating the effects of chronic exposure of pancre-atic islets and beta cell lines to sulfonylureas in vitro have shown that the desensitization in certain cases

is not limited to subsequent drug-induced insulin

Keywords

beta cells; metabolism; sulfonylurea

Correspondence

L Brennan, UCD School of Biomolecular

and Biomedical Science, UCD Conway

Institute, University College Dublin, Belfield,

Dublin 4, Ireland

Fax: +353 1 2837211

Tel: +353 1 7166781

E-mail: lorraine.brennan@ucd.ie

(Received 9 June 2006, revised 21

Septem-ber 2006, accepted 22 SeptemSeptem-ber 2006)

doi:10.1111/j.1742-4658.2006.05513.x

Prolonged exposure of pancreatic beta cells to the sulfonylureas glibenca-mide and tolbutaglibenca-mide induces subsequent desensitization to the actions of these drugs The precise mechanisms underlying this desensitization remain unknown, prompting the present study, which investigated the impact of prolonged sulfonylurea exposure on glucose and energy metabolism using clonal pancreatic BRIN-BD11 beta cells Following prolonged exposure to tolbutamide, BRIN-BD11 beta cells were incubated in the presence of [U-13C]glucose, and isotopomer analysis revealed that there was a change

in the ratio of flux through pyruvate carboxylase (EC 6.4.1.1) and pyruvate dehydrogenase (EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4) Energy status in intact BRIN-BD11 cells was determined using 31P-NMR spectroscopy Exposure to tolbutamide did not alter the nucleotide triphosphate levels Collectively, data from the present study demonstrate that prolonged expo-sure of beta cells to tolbutamide results in changes in flux through key enzymes involved in glucose metabolism that, in turn, may impact on glucose-induced insulin secretion

Abbreviations

KATPchannel, ATP-sensitive potassium ion channel; NTP, nucleotide triphosphate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase.

secretion, but may also affect nutrient-induced insulin

secretion [10–13] The precise mechanisms underlying

beta cell drug-induced desensitization remains elusive,

and it remains unclear as to whether it results from

depletion of insulin stores or functional changes in

signalling pathways [14] In a study investigating

desensitization due to chronic exposure to

glibenca-mide in MIN6 cells, a decrease in the number of

fun-ctional KATP channels on the plasma membrane was

observed [15] This would support a homologous

desensitization theory, where the primary cause of the

desensitization is due to the occupancy of receptor

sites and subsequent modification of the downstream

pathways [14] However, other studies have

demon-strated that prolonged treatment with sulfonylurea

drugs affects the subsequent secretory response to

other stimuli [12,13,16], indicating the presence of

heterogeneous desensitization

Acute stimulation of insulin secretion by glucose

involves metabolism via oxidative and anaplerotic

pathways involving pyruvate dehydrogenase (PDH;

EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4) and pyruvate

carboxylase (PC; EC 6.4.1.1) [17–19] The resultant

increase in ATP concentration closes the KATP

chan-nels, resulting in membrane depolarization, opening

of the voltage-dependent Ca2+ channels, Ca2+ influx,

and a subsequent increase in cytosolic Ca2+ ([Ca2+]c)

which in turn triggers insulin secretion Additionally,

the influx of Ca2+results in an uptake of Ca2+by the

mitochondria, which may activate mitochondrial

meta-bolism and result in further increases in ATP

concen-tration [20–22] The present study investigated the

effects of prolonged exposure to sulfonylurea drugs on

beta cell metabolism using a combination of different

NMR techniques, and utilizing the well-characterized

clonal glucose-responsive pancreatic beta cell line,

BRIN-BD11 [23,24]

Results

Metabolism of [1-13C]glucose in the presence and

absence of sulfonylurea(s)

Following 18 h of prior culture in the presence of

tol-butamide or glibencamide, the cells were incubated for

1 h in the presence of [1-13C]glucose with or without

the sulfonylurea drug The main metabolites produced

included glutamate labelled at positions C2, C3 and

C4, lactate and alanine labelled at position C3, and

aspartate labelled at positions C2 and C3 Comparison

of the label distribution between the different

treat-ments indicated that there was no significant change

in the total concentration of glutamate labelled at

position C4 (Table 1) In the case of tolbutamide pre-treatment, there was a general trend for decreased labelling of glutamate in positions C2 and C3, but the changes were significant only for the C2 position The total 13C concentration of alanine and lactate labelled

at position C3 did not change significantly However,

in the presence of tolbutamide, the total amount of

13C label present in aspartate C2 and C3 increased significantly The percentage enrichments of the glutamate peaks in the presence and absence of the drugs are reported in Table 2 No significant changes were observed for glutamate C3 and C4 In the presence of tolbutamide, there was a decrease in the percentage enrichment for glutamate C2 The amount

of glucose remaining in the medium was measured in the presence and absence of drugs, and was found to

be significantly different only in the presence

of glibencamide; under control conditions, the amount

of glucose remaining was 19.5 ± 1.6 lmolÆmg)1 protein, whereas in the presence of glibencamide, the concentration was 14.3 ± 0.9 lmolÆmg)1 protein (P < 0.005) The concentration of glucose remaining

in the medium after tolbutamide exposure was 17.9 ± 2.1 lmolÆmg)1 protein The insulin released into the medium at the end of a 1 h incubation period following the preculture with and without the drugs

Table 1 Amount of 13 C-labelled amino acids present after incuba-tion for 1 h with [1-13C]glucose in the presence and absence of sul-fonylurea drugs Values are given as nmol per mg protein ± SD (n ¼ 3) All experiments were carried out after a preincubation per-iod of 18 h in the presence or absence of drug.

Control + Tolbutamide + Glibencamide

*P < 0.05.

Table 2 Percentage enrichment in the glutamate carbons following incubation for 1 h with [1- 13 C]glucose in the presence and absence

of sulfonylurea drugs Values are expressed as a percentage ± SD (n ¼ 3) All experiments were carried out after a preincubation period of 18 h in the presence or absence of drug.

Control + Tolbutamide + Glibencamide

*P < 0.05.

was measured Preincubation in the presence of the

drugs significantly reduced the amount of insulin

released (Fig 1)

Effects of tolbutamide and glibencamide on

[U-13C]glucose metabolism

To probe further the effect of tolbutamide and to

investigate the observed increase in aspartate

produc-tion, flux analysis was carried out using [U-13

C]glu-cose Following prior culture for 18 h in the presence

of tolbutamide, BRIN-BD11 cells were incubated in

the presence of tolbutamide and [U-13C]glucose for

2 h Control experiments were run in parallel with no

drug present A typical spectrum obtained from

con-trol cellular extracts is shown in Fig 2 There was no

significant change in the total amount of labelled

glu-tamate, lactate or alanine produced following culture

with tolbutamide (data not shown) The ratio of the

flux through PC and PDH was determined in the

pres-ence and abspres-ence of the drug, as described in Experi-mental procedures (Table 3) A significant reduction in the ratio was found in the presence of tolbutamide The fraction of acetyl-CoA derived from [U-13 C]glu-cose was determined, and is reported in Table 4 Preincubation with tolbutamide did not change the percentage significantly, indicating that the fraction of glucose entering the tricarboxylic acid cycle via the PDH-mediated conversion to acetyl-CoA did not

0

1

2

3

4

5

6

7

Fig 1 Effects of culture (18 h) with 100 l M tolbutamide (Tol) or

1 l M glibencamide (Glib) on glucose-induced insulin release over a

subsequent 1 h incubation period Values are mean ± SD Control

conditions refer to culture and subsequent incubation in the

absence of a drug *P < 0.05.

Glucose

80 70 60 50 40 30 20

Chemical Shift (p.p.m.)

Fig 2 A typical 13 C-NMR spectrum obtained for control conditions after incubation in the presence of 8.4 m M [U- 13 C]glucose for 2 h Lac, lactate.

Table 3 Ratio of flux through pyruvate carboxylase (PC) and pyru-vate dehydrogenase (PDH) calculated from C2 and C4 resonances

of glutamate in the presence and absence of a sulfonylurea drug For all experiments, cells were incubated in the presence of [U-13C]glucose for 2 h following 18 h of prior culture in the presence

or absence of the drug Values are expressed as averages ± SD.

*P < 0.05.

Table 4 Percentage of acetyl-CoA derived from [U-13C]glucose fol-lowing incubation in the presence and absence of a sulfonylurea drug Values are expressed as a percentage ± SD Cells were incu-bated in the presence of [U-13C]glucose with or without a drug for

2 h following a preincubation period of 18 h in the presence or absence of a drug.

Condition

Percentage labelled from [U- 13 C]glucose

change Thus, the observed change in the ratio of flux

through PC and PDH must have been due to changes

in anaplerotic metabolism through PC

When cells were preincubated in the presence of

glibencamide followed by [U-13C]glucose, the ratio of

the flux through PC and PDH did not differ

signifi-cantly from the control value

31P-NMR studies of intact pancreatic BRIN-BD11

beta cells

A typical spectrum obtained from cells grown on

fibra-cel beads in a mini-bioreactor is shown in Fig 3 The

T1 (longitudinal relaxation) values for the NTP peaks

were calculated using inversion recovery experiments

The following values were found: a, 0.65 ± 0.14 s;

b, 0.55 ± 0.05 s; and c, 0.88 ± 0.19 s Control

condi-tions represent condicondi-tions where cells were maintained

in standard RPMI-1640 medium supplemented with

2 mm glutamine, 10% (v⁄ v) fetal bovine serum, and

0.1% antibiotics When tolbutamide was added to the

culture medium to give a final concentration of

100 lm, there were no significant changes in the

nuc-leotide triphosphate (NTP) levels over a 24 h period

(Fig 4) The free cytoplasmic ADP concentration is

directly proportional to the phosphocreatine⁄ ATP

ratio [25,26] There was no significant change in this

ratio on addition of tolbutamide, which, in the absence

of a change in the intracellular pH or total creatine

pool, indicated that there was no change in free ADP

concentration

Discussion

Previous studies using clonal pancreatic BRIN-BD11

beta cells have demonstrated that prolonged exposure

(18 h) to sulfonylureas induces specific and readily reversible desensitization to subsequent treatment with the drugs [13,27] It has also been reported that chro-nic exposure (72–144 h) can result in an irreversible concentration- and time-dependent decline in sulfonyl-urea-induced insulin secretion [28] As the mechanisms underlying the latter effects remain to be determined, the principal aim of this study was to investigate the impact of sulfonylurea exposure on cellular metabo-lism using clonal pancreatic BRIN-BD11 beta cells Under the experimental conditions used in this study, there was a significant decrease in acute insulin release following preincubation with the drugs compared to control conditions Previous studies on tolbutamide have demonstrated that under the same conditions as described in this study, the insulin content does not change [27]

Prolonged exposure to tolbutamide and glibenca-mide did not significantly alter the amount of glu-tamate labelled at positions C4 and C3 following a

1 h incubation with [1-13C]glucose The amount of

Chemical Shift (p.p.m.)

20 10 0 -10 -20

NTP

Fig 3 A typical 31 P-NMR spectrum of intact BRIN-BD11 cells grown in the mini-bioreactor MDP, methylene diphosphonate; PME, phospho-monoesters; Pi, inorganic phosphate; PCR, phosphocreatine; NTP, nucleotide triphosphate.

0 0.1 0.2 0.3 0.4 0.5 0.6

0 5 10 15 20 25 30 35 40

control tolbutamide

time (h) Fig 4 Nucleotide triphosphate levels in intact BRIN-BD11 cells from a representative culture of cells After a control period of

15 h, the cells were perfused in medium with 100 l M tolbutamide for 24 h.

label at the C2 position showed a small decrease

fol-lowing incubation in the presence of tolbutamide

The amount of labelled lactate and alanine did not

significantly change following incubation with either

drug However, following prolonged exposure to

tol-butamide, the amount of aspartate labelled at

posi-tions C2 and C3 significantly increased Aspartate

can be formed via a transamination reaction with

oxaloacetate, which is itself produced from pyruvate

via a reaction catalysed by PC Oxaloacetate is in

equilibrium with malate, which can leave the

mitoch-ondrial matrix and in the cytosol is converted back

to pyruvate via malic enzyme (pyruvate cycling) To

gain a better understanding of these changes,

experi-ments were subsequently performed using [U-13

C]glu-cose, and analysis of the isotopomers formed allowed

calculation of the fluxes through specific enzymes

(Fig 5)

A decrease in the ratio of fluxes through PC and

PDH (PC⁄ PDH) was determined following 18 h of

exposure to tolbutamide However, there was no

change in the fraction of acetyl-CoA labelled from

[U-13C]glucose via the PDH pathway, indicating that

the change in ratio was attributable to a reduction in

the flux through PC Notably, other studies

investi-gating the acute effects of glibencamide and

megliti-nide on glucose oxidation in mouse pancreatic islets

found no inhibitory effects, consistent with our

obser-vations that oxidative pathways of glucose metabolism

remain unchanged [29] In recent years, the importance

of flux through anaplerotic pathways in beta cells has

been highlighted Cline et al [30] reported a strong

positive correlation between insulin secretion and PC

flux in INS-1 cells Furthermore, Lu et al

demonstra-ted that the responsiveness of INS-1 cells to glucose-stimulated insulin secretion was linked to the cycling [31] of pyruvate (i.e the flux through the pyruvate– malate and pyruvate–citrate cycles) In a recent study, Fransson et al showed, by use of a PC inhibitor, the importance of flux through PC for responsiveness to glucose [32] In the present study, the observed decrease in flux through PC observed after prolonged exposure to tolbutamide may be a contributary factor

to the resulting drug-induced desensitization to acute stimulation by glucose

The present data also indicate differential effects of the two sulfonylureas on beta cell metabolism, as glib-encamide did not change end-product concentrations

or the ratio of fluxes through PC and PDH Preincu-bation with either of the drugs resulted in reduction of subsequent glucose-stimulated insulin release, suggest-ing that there is not a common mechanism of desensi-tization However, in the present studies, a relatively low concentration of glibencamide (1 lm) was used, due to the reported higher affinity of this drug for

KATP channels [33] compared to tolbutamide Glyb-encamide at 1 lm induces a similar secretory response

to that observed for 100 lm tolbutamide [16,34,35] Early studies investigating the effects of sulfonyl-ureas on ATP levels reported reduced concentrations

in islets [36–38] More recently Elmi et al [29] showed that glibencamide reduced ATP concentration in the absence of glucose, but the effects were not observed

at 10 mm glucose These observations were used to suggest that the reduced ATP levels may result from increased consumption of ATP by activation of the

Na+⁄ K+pump This is consistent with the results of a

31P-NMR study of intact b-HC9 cells, which revealed

OAA

citrate

malate

pyruvate

Acetyl-CoA PC

PDH

PC aspartate

1 2 3 4 5

Fig 5 Overview of the metabolism of [U- 13 C]glucose For simplicity, only the iso-topomers of glutamate formed after one turn of the tricarboxylic acid cycle (TCA) are shown The filled circles represent labelling

of the carbon position with 13 C [U- 13 C]Pyru-vate enters the TCA cycle via pyruC]Pyru-vate dehydrogenase (PDH), forming [1,2- 13 C]acetyl-CoA and consequently [4,5-13C]glutamate If pyruvate enters via pyruvate carboxylase (PC), two 13 C isotopo-mers of oxaloacetate are derived,

[1,2,3-13C]oxaloacetate and [3,4-13 C]oxalo-acetate, consequently leading to [2,3- 13 C]glutamate and [1,2,3- 13 C]glutamate [48] Glu, glutamate; OAA, oxaloacetate; a-KG, a-ketoglutarate.

that ATP levels did not change on addition of the

sul-fonylurea glyburide at glucose concentrations of 5 mm

[39] However, another study using MIN-6 cells

showed that if the cells were initally primed with

tol-butamide (200 lm), the subsequent increase in ATP in

response to 30 mm glucose was potentiated [40] All of

these studies are distinct from ours, in that they

focused on the effects of acute sulfonylurea exposure,

whereas we examined the effects of prolonged exposure

on ATP levels using31P-NMR Our data are consistent

with other studies, which reported no alteration in

cel-lular ATP levels after addition of sulfonylureas in the

presence of glucose

Collectively, the present data demonstrate novel

changes to fluxes in glucose metabolism following

pro-longed exposure to the important sulfonylurea drug

tolbutamide, resulting in a 25% decrease in the

PC⁄ PDH ratio Furthermore, these data reveal a

reduction in anaplerotic flux through PC These

obser-vations are notable in that they raise the possibility

that chronic sulfonylurea exposure in vivo may impact

on glucose metabolism, which may contribute to the

reported phenomena of sulfonylurea desensitization

and, indeed, sulfonylurea failure in type 2 diabetes

Future studies determining the molecular mechanisms

of tolbutamide-mediated reduction in flux through PC

may lead to the design of more effective insulinotropic

drugs in the future

Experimental procedures

Reagents

d-[1-13C]Glucose and d-[U-13C]glucose were obtained from

Goss Scientific (Great Baddow, Essex, UK) All other

chemicals were obtained from Sigma-Aldrich Chemical

Company (Poole, Dorset, UK) Culture media and fetal

bovine serum were obtained from Gibco (Glasgow, UK)

Treatment of BRIN-BD11 cells with drugs

Pancreatic BRIN-BD11 beta cells were utilized in these

studies [41], representing a particularly useful model in

which to conduct extensive NMR studies [23,24]

BRIN-BD11 cells were maintained in RPMI-1640 tissue culture

medium with 10% (v⁄ v) fetal bovine serum, 0.1%

antibiot-ics (100 UÆmL)1 penicillin and 0.1 mgÆmL)1 streptomycin)

and 11.1 mm d-glucose (pH 7.4) The cells were maintained

at 37C in a humidified atmosphere of 5% CO2and 95%

air using a Forma Scientific (Waltham, MA) incubator For

experiments on prolonged exposure to sulfonylureas, the

cells were grown in T175 flasks and treated for 18 h in

the presence of the drug at the specified concentration

(tolbutamide 100 lm and glibencamide 1 lm) Cells were then washed with NaCl⁄ Pi and preincubated at 37C for

20 min in Krebs⁄ Ringer bicarbonate buffer with 1.1 mm

d-glucose (115 mm NaCl, 4.7 mm KCl, 1.28 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4.7H2O, 10 mm NaHCO3,

5 gÆL)1 BSA, pH 7.4) This was followed by incubation in the presence of 8.4 mm labelled glucose ([1-13C]glucose or [U-13C]glucose) and drug for a specified period (1 h or 2 h) Control experiments were carried out in parallel in the absence of the drug

Following the incubation period, the medium was removed and stored at ) 20 C Subsequently, the glucose concentration and the insulin released were measured The insulin released was measured using a Mercodia (Uppsala, Sweden) ultrasensitive rat insulin ELISA The metabolites were extracted using a perchloric acid extraction procedure Briefly, the cells were washed with ice-cold NaCl⁄ Pi Per-chloric acid (6%) was added, and the cells were scraped off the culture flasks The extracts of six culture flasks (approximately 108cells) were pooled and centrifuged at

200 g with a Sigma 2-15 rotor The resulting supernatant was neutralized with KOH (5 m and 0.1 m solutions), and the pellets were soaked overnight in 0.1 m NaOH The pro-tein concentration was determined using the Lowry method [42] The neutralized supernatant was centrifuged (200 g with a Sigma 2-15 rotor), and the supernatant was treated with Chelex-100 resin and then lyophilized Each experi-ment was carried out on at least two independent cultures

of the BRIN-BD11 cells The lyophilized cell extracts were dissolved in 3 mL of potassium phosphate buffer (100 mm,

pH 7.0) and then centrifuged at 200 g with a Sigma 2-15 rotor The supernatant was carefully removed, 10% D2O was added, and the pH was checked and adjusted when necessary with 0.1 m NaOH and 0.1 m HCl NMR experi-ments were performed as detailed below

Culture of BRIN-BD11 cells at high density in a mini-bioreactor

A system was set up to monitor energy metabolism in intact BRIN-BD11 cells as previously described [43] Briefly, it consists of a 2 L stirred tank fermenter in which the temperature, pH, CO2content and dissolved O2content

of the culture medium were monitored and maintained within a specified range The mini-bioreactor is a purpose-built bioreactor consisting of a capped polysulfone tube (10 mm diameter) with an inlet tube and an outlet tube Cells were maintained attached to fibracel disks (New Brunswick Scientific, Edison, NJ) in the mini-bioreactor and were perfused with medium from the fermenter, which was pumped to the mini-bioreactor in the NMR magnet via water-jacketed tubing at 37C Medium in the

ferment-er was replaced by fresh medium using a ‘feed and bleed’ system By adjustment of the feed and bleed system,

nutrients can be maintained at a certain level For seeding

of the bioreactor, the cells were pumped into the

mini-bio-reactor as a slow rate over a period of 2 h Typically

1· 108 to 3· 108cells were passed through the carriers,

and attachment of 85–90% of the cells was routinely

found

NMR spectroscopy

13C-NMR of perchloric acid extracts

For 13C-NMR experiments, an insert containing 5% v⁄ v

dioxane in water was used as an external signal intensity

reference A solution of l-alanine, l-glutamate, lactate and

d-glucose, each at a concentration of 100 mm, was prepared

and used to quantitate concentrations of metabolites in the

13

C spectra Proton-decoupled13C spectra were acquired on

a Bruker (Karlsruhe, Germany) DRX 500 spectrometer

using a 10 mm broadband probe Typically, spectra were

acquired with 32 000 data points using 90 pulses,

260 p.p.m spectral width, 2.5 s relaxation delay, and

12 000–20 000 scans Spectra were recorded at 25C

Chemical shifts were referenced to tetramethylsilane at

0 p.p.m Data were processed with no zero filling using

Bruker winnmr software, and exponential multiplications

with 2 Hz line broadening were performed The

assign-ments of the intermediate metabolites were made by

com-parison with chemical shift tables in the literature [44] or

by addition of 100 mm unlabelled amino acid The amount

of 13C in each resonance was evaluated by integration of

the extract peaks and the corresponding peaks in the

stand-ard sample relative to the dioxane signal Corrections for

the natural abundance signal were made In the case of the

aspartate peaks, the amount of13C was estimated by use of

the integrals and the known dioxane concentration The

contributions of the individual isotopomers were assessed

using the deconvolution routines in winnmr The absolute

enrichments of the l-glutamate were related to the

glutam-ate concentration in the extracts, determined by enzymatic

methods, to give the specific enrichments [45] The fluxes

reported were obtained by analysis of the isotopomers of

glutamate C2 and C4 The ratio between flux through PC

and PDH was calculated as follows ([2,3-13C2] +

[1,2,3-13C3])⁄ [4,5-13C2] + [3,4,5-13C3] [46] The fraction of

acetyl-CoA labelled from [U-13C]glucose was calculated

using the following equation for the C4 peak:

(3,4,5-13C)· C4 ⁄ C3 [47]

31P-NMR of intact cells

Following removal of the upper barrel of the magnet, the

mini-bioreactor was carefully guided into the probe head

for the collection of NMR data with the aid of a

custom-built holder Typically, spectra were acquired with 8000

data points using 90 pulses, 50 p.p.m spectral width, 2.0 s

relaxation delay, and 512 scans Spectra were recorded at

37C Chemical shifts in aqueous media were referenced

to methylene diphosphonate at 17.0 p.p.m., which was contained in a sealed capillary in the mini-bioreactor Expo-nential multiplications with 20 Hz line broadening were performed using winnmr software The three phosphate groups in NTPs give three distinct peaks These peaks were primarily attributed to ATP The T1 values of the NTP peaks were determined using inversion recovery experiments

in three independent sets of cells, and these values were then used to optimize the NMR acquisition parameters To investigate the effects of tolbutamide on energy metabolism, the cells were perfused with medium containing 100 lm tol-butamide for a period of 24 h Spectra were recorded every

40 min In some cases, slight acidification of the bioreactor environment was apparent by appearance of a shoulder on the Pi peak attributed to intracellular Pi When this occurred, the flow rate through the bioreactor was increased as previously described [43]

Statistical analysis The results are presented as mean ± SD for n separate determinations Groups of data were compared using Student’s unpaired t-test Differences were considered signi-ficant at P < 0.05

Acknowledgements

LB was in receipt of a Health Research Board of Ireland postdoctoral fellowship, which is gratefully acknowledged (PD⁄ 2002 ⁄ 9)

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