Acute insulin-releasing actions of amino acids have been studied in detail, but comparatively little is known about the β-cell effects of long-term exposure to amino acids. The present study examined the effects of prolonged exposure of β-cells to the metabolizable amino acid L-alanine. Basal insulin release or cellular insulin content were not significantly altered by alanine culture, but acute alanine-induced insulin secretion was suppressed by 74% (P<0.001). Acute stimulation of insulin secretion with glucose, KCl or KIC (2-oxoisocaproic acid) following alanine culture was not affected. Acute alanine exposure evoked strong cellular depolarization after control culture, whereas AUC (area under the curve) analysis revealed significant (P<0.01) suppression of this action after culture with alanine. Compared with control cells, prior exposure to alanine also markedly decreased (P<0.01) the acute elevation of [Ca2+]i (intracellular [Ca2+]) induced by acute alanine exposure. These diminished stimulatory responses were partially restored after 18 h of culture in the absence of alanine, indicating reversible amino-acid-induced desensitization. 13C NMR spectra revealed that alanine culture increased glutamate labelling at position C4 (by 60%; P<0.01), as a result of an increase in the singlet peak, indicating increased flux through pyruvate dehydrogenase. Consistent with this, protein expression of the pyruvate dehydrogenase kinases PDK2 and PDK4 was significantly reduced. This was accompanied by a decrease in cellular ATP (P<0.05), consistent with diminished insulin-releasing actions of this amino acid. Collectively, these results illustrate the phenomenon of β-cell desensitization by amino acids, indicating that prolonged exposure to alanine can induce reversible alterations to metabolic flux, Ca2+ handling and insulin secretion.
- amino acid
- insulin secretion
- intracellular calcium
- pancreatic β-cell
- pyruvate dehydrogenase kinase (PDK)
Insulin secretion from pancreatic β-cells is tightly regulated by circulating glucose and is modulated by a number of physiological and pharmacological factors [1–4]. Amino acids represent an important class of modulators of pancreatic β-cell function, and specific individual or mixtures of amino acids regulate insulin secretion both in vitro and in vivo. Pancreatic β-cells are equipped with a range of specific amino acid transporters, many of which are Na+-dependent [5,6]. Three principal modes of insulinotropic action of amino acids have been characterized [7,8]. First, direct membrane depolarization resulting from β-cell transport of cationic amino acids (such as arginine) promotes Ca2+ channel opening and insulin release. Other metabolizable amino acids (such as leucine) can increase intracellular ATP evoking the closure of KATP channels (ATP-sensitive K+ channels), membrane depolarization and Ca2+ influx stimulating insulin release. In addition to these mechanisms, certain amino acids including alanine may stimulate insulin release through both metabolism and as a direct result of the membrane-depolarizing actions of Na+ co-transport triggering Ca2+ influx and ultimately insulin release. Although the insulinotropic effects of arginine and leucine have long been known [9–11], the importance of alanine in the acute regulation of β-cell metabolism and function has only been appreciated more recently [12–14]. Of particular note is the positive effect of the abundant amino acid alanine on glucose metabolism and insulin secretion , suggesting that this amino acid may have a significant impact on β-cells.
Although acute exposure to nutrients and other insulinotropic agents exert positive β-cell actions, prolonged exposure may induce desensitization and other detrimental effects [1,3,4,15]. Glucose is the principal physiological regulator of insulin release, but chronic exposure to high levels of this sugar is associated with β-cell deterioration, so-called glucose desensitization and glucotoxicity, perhaps involving oxidative stress [16–18], but this has been questioned [19,20]. Similarly, non-esterified fatty acids (‘free’ fatty acids) act as important signalling molecules and β-cell fuels, enhancing insulin release, but long-term exposure can induce β-cell lipotoxicity, particularly in the presence of high glucose levels . A recent metabolomic analysis of urine from Type 2 diabetic subjects and animals models has shown that the main metabolic disturbances occurred in amino acid metabolism, with an increased excretion of glutamine, glutamate, alanine, taurine and ornithine in human subjects . The long-term consequences of such altered amino acid concentrations on the pancreatic β-cell has received little attention to date.
A previous study indicated that prolonged exposure to the amino acid alanine induced the up-regulation of gene expression of certain metabolic and signal transduction elements coupled with enhanced protection against pro-inflammatory cytokine-induced apoptosis . Notably, these preliminary studies indicated an alteration in β-cell responsiveness to subsequent amino-acid-induced stimulation . This observation prompted the present study examining in more detail the nature of the demise in insulin secretion and β-cell function following prolonged alanine exposure, demonstrating induction of reversible alterations to metabolic flux, Ca2+ handling and insulin secretion.
MATERIALS AND METHODS
L-[3-13C]Alanine was obtained from Goss, and 125I-bovine insulin was purchased from Amersham Biosciences. All other chemicals were obtained from Sigma and BDH chemicals. Culture medium and FBS (foetal bovine serum) were obtained from Gibco. The membrane potential kit and calcium assay kit were purchased from Molecular Devices.
Cell culture and treatment with L-alanine for NMR studies
Experiments utilized glucose- and amino-acid-responsive clonal pancreatic BRIN-BD11 β-cells [4,24]. These cells have proven to be particularly useful as model β-cells for studies involving NMR-based experiments which require substantial cellular mass [12,25]. The origin of BRIN-BD11 cells is described elsewhere [4,26]. These cells provide an appropriate β-cell model as shown by studies of insulin secretion [4,24], electrophysiology, Ca2+ handling  and cellular defence . In addition, signalling, insulin secretory and cell viability responses to glucose and amino acids, as well as other stimuli, have been well-characterized [26–29].
BRIN-BD11 cells were grown and maintained in RPMI 1640 tissue culture medium with 10% (v/v) FBS, 0.1% antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin) and 11.1 mmol/l D-glucose at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, as described previously .
For experiments investigating prolonged exposure to L-alanine, cell monolayers were maintained in T175cm2 flasks (Greiner) and were treated for 18 h in the presence of 10 mmol/l L-alanine. A concentration of 10 mmol/l was used as this provides a robust and reproducible stimulus for insulin secretion experiments . For comparison, cells were grown in parallel in the absence of L-alanine (standard culture; Control). Cells were then washed with PBS and pre-incubated at 37 °C for 20 min in KRBB (Krebs–Ringer bicarbonate buffer) comprising 115 mmol/l NaCl, 4.7 mmol/l KCl, 1.28 mmol/l CaCl2, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4·7H2O, 10 mmol/l NaHCO3 and 5 g/l BSA (pH 7.4) supplemented with 1.1 mmol/l D-glucose. This was followed by incubation for 1 h with test KRBB containing 1.1 mmol/l D-glucose and 10 mmol/l L-[3-13C]alanine, and aliquots of the test buffer were removed and stored at −20 °C for further analysis. After test exposures, metabolites were extracted from cells using a perchloric acid extraction procedure. Briefly, cells were washed with ice-cold PBS, perchloric acid (6%, v/v) was added and cells were scraped from tissue culture flasks. Extracts of six culture flasks (approx. 1×108 cells) were pooled, centrifuged at 200 g and the resulting supernatants were neutralized with KOH (5 mol/l and 0.1 mol/l solutions) before soaking the pellets overnight in 0.1 mol/l NaOH. The protein concentration was determined using the Lowry method , the neutralized supernatant was centrifuged again (at 200 g), and the supernatant was treated with a Chelex-100 resin before freeze-drying. Each experiment was carried out on at least three independent cultures of the BRIN-BD11 cells. Freeze-dried cell extracts were dissolved in 3 ml of potassium phosphate buffer (100 mmol/l; pH 7.0), centrifuged and the supernatant was carefully removed before the addition of 10% 2H2O and the pH checked and adjusted where necessary with 0.1 mol/l NaOH or 0.1 mol/l HCl. NMR analyses were then performed as detailed below.
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 mmol/l, was prepared and used to quantify the concentrations of metabolites in the 13C spectra. Proton-decoupled 13C spectra were acquired on a Bruker DRX 500 spectrometer using a 10 mm broadband probe. Typically, spectra were acquired with 32 K data points using 90° pulses, a 260 p.p.m. spectral width, a 2.5 s relaxation delay and 12000–20000 scans. Spectra were recorded at 25 °C. 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. Assignments of intermediate metabolites were made by comparison with chemical shift tables in the literature or by the addition of 100 mmol/l of unlabelled amino acid. The amount of 13C in each resonance was evaluated by integration of the extract peaks and the corresponding peaks in the standard sample relative to the dioxane signal. Corrections for the natural abundance signal were made. In the case of the aspartate peaks, the amount of 13C was estimated by the use of the integrals and the known dioxane concentration. The contribution of the individual isotopomers was assessed using the deconvolution routines in WINNMR. The absolute enrichments of L-glutamate were related to the glutamate concentration in the extracts to give the specific enrichments.
Determination of insulin release and cellular content
Insulin release was determined from BRIN-BD11 cell monolayers. Briefly, cells were harvested, resuspended in culture medium and seeded in each well of 24-well multiplates at a density of 1.5×105 cells/well. After overnight incubation and attachment at 37 °C, standard RPMI 1640 tissue culture medium was replaced with standard culture medium (no L-alanine) or culture medium supplemented with 10 mmol/l L-alanine, and cells were returned to culture. After 18 h, the culture medium was removed from each well and replaced with 1 ml of KRBB supplemented with 0.1% BSA and 1.1 mmol/l D-glucose for a 20 min pre-incubation at 37 °C. At the end of the pre-incubation period, pre-incubation KRBB was removed and replaced with 1 ml of test KRBB supplemented with 1.1 mmol/l D-glucose and 10 mmol/l L-alanine, as indicated in the Figures. After incubation for 1 h, aliquots of test buffer were removed from each well and stored at −20 °C. Cellular insulin was extracted by the addition of 1 ml of ice-cold acid/ethanol solution [75% (v/v) ethanol and 1.5% (v/v) concentrated HCl]. Insulin was measured from each sample by a dextran-charcoal RIA , using guinea-pig anti-porcine insulin serum and a rat insulin standard.
Assessment of cellular integrity following alanine culture
Following culture in the absence or presence of alanine, cellular integrity was assessed by means of either the Neutral Red or MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. For the Neutral Red assay, 2×104 cells were seeded into each well of 96-well plates and cultured for 18 h in the absence or presence of 10 mmol/l L-alanine. Cells were then exposed to 100 μl of Neutral Red solution (PBS containing 0.05 mg/ml Neutral Red) for 2 h and, after washing with PBS, 100 μl of acetic acid solution (50 ml of ethanol, 1 ml of acetic acid and 49 ml of distilled H2O) was added to each well and the plates were shaken for 15 min for cell lysis and Neutral Red release from the cells. Absorbance from each well was then recorded at 540 nm and compared with control (culture in absence of 10 mmol/l L-alanine), and viability was expressed as a percentage relative to control. For MTT analyses, 2×104 cells were seeded into each well of 96-well plates and cultured for 18 h in the absence or presence of 10 mmol/l L-alanine before incubation with 100 μl of MTT solution (1:10 dilution of the 5 mg/ml stock of MTT in RPMI without Phenol Red) at 37 °C for 30 min. The MTT solution was then removed and the cells were lysed with 100 μl of DMSO. Absorbances were recorded at 562 nm with a reference wavelength of 650 nm, and cell viability was expressed as a percentage relative to control.
Determination of ATP and protein expression
For ATP and protein measurements, the cells were seeded into six-well plates (1×106 cells) and were allowed to form monolayers overnight. Cells were then exposed to culture in the absence or presence of 10 mmol/l L-alanine for 18 h, followed by incubation for 1 h in KRBB, as described above. Following incubation, cells were lysed with a somatic cell ATP-releasing reagent and placed on ice . Intracellular ATP was measured using a luciferin/luciferase-based assay (Biaffin) on ice .
Protein expression was examined using Western blotting, where cells were washed with ice-cold PBS before lysis in RIPA buffer [0.5 mol/l Tris/HCl (pH 7.4), 1.5 mol/l NaCl, 2.5% deoxycholic acid, 10% Nonidet P40 and 10 nmol/l EDTA]. Samples were centrifuged at 12000 g for 15 min at 4 °C, the supernatant was collected and the total protein was determined using a bicinchoninic acid assay. Samples were then subjected to SDS/PAGE using a 12.5% (w/v) resolving gel, after which the resolved proteins were transferred on to nitrocellulose membranes and blocked for 1 h at room temperature (20 °C) in Tris-buffered saline supplemented with 0.1% Tween and 5% (w/v) non-fat powdered milk. Nitrocellulose blots were incubated for either 2 h with the PDK [PDH (pyruvate dehydrogenase) kinase]-2 polyclonal antibody (Abgent) or 4 h with the PDK-4 polyclonal antibody, washed and incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies (Santa Cruz Biotechnology). Bound antibody was visualized using enhanced chemiluminescence according to the manufacturer's instructions (Pierce). Equal loading was determined by analysis of total JNK (c-Jun N-terminal kinase) or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The blots were then exposed to Hyperfilm and the bands were quantified by scanning densitometry.
Semi-quantitative analyses of PDK-2 and PDK-4 gene expression
For semi-quantitative detection of PDK-2 and PDK-4 mRNA levels, cells were grown and maintained in six-well plates. Total RNA was extracted using the thiocyanate/phenol/chloroform method using TRI Reagent, according to the manufacturer's protocol (Molecular Research Centre Inc.). cDNA was synthesized and amplified by PCR using primers for PDK-2, PDK-4 and 18S (the housekeeping gene). The following primer sequences were used: PDK-2, forward 5′-AGTTCACTGATGCCCTGGTC-3′ and reverse 5′-GTTGGTGGCATTGACTTCCT-3′; PDK-4, forward 5′-CGTCGCCAGAATTAAAGCTC-3′ and reverse 5′-TAACCAAAACCAGCCAAAGG-3′. PCR was carried out according to the following protocol: a 2 min denaturing step at 94 °C, followed by 25–30 cycles of amplification (45 s of denaturation at 94 °C, 45 s of annealing at 60 °C and 45 s of extension at 72 °C) and a final extension step of 2 min at 72 °C. PCR products were analysed on ethidium-bromide-stained agarose gels, photographed under UV transillumination and the results are expressed relative to the housekeeping gene 18S.
Determination of metabolite levels
Alanine concentrations were determined in the medium following the incubation periods using an enzymatic-based reaction coupled to the production of NADH. Total glutamate and lactate levels were determined using a YSI 7100 amino acid analyser.
Measurement of membrane potential and [Ca2+]i (intracellular [Ca2+])
Membrane potential and [Ca2+]i were determined using monolayers of BRIN-BD11 cells, as described previously . Cells were seeded into 96-well black-walled clear-bottom microplates at a density of 1.0×105 cells/well and allowed to attach overnight before replacing the culture medium with either standard culture medium or culture medium supplemented with 10 mmol/l L-alanine. After culture for 18 h, the medium in each well was replaced with 100 μl of KRBB and monolayers were incubated for 20 min, after which 100 μl of either Flex membrane potential assay kit reagent  or Flex calcium assay kit reagent (Molecular Devices) was added to the wells at 37 °C, as described previously . Fluorometric data were acquired using the FlexStation® scanning fluorimeter and integrated fluid transfer workstation (Molecular Devices). The cells were exposed to excitation from a xenon-arc flashlamp at a wavelength of 530 nm (membrane) or 485 nm ([Ca2+]i), and subsequent fluorescence emission was measured at 565 nm (membrane) or 525 nm ([Ca2+]i) using a bottom read mode. Emission cut-off filters were set at 550 nm for membrane potential or 515 nm for [Ca2+]i.
Results are expressed as means±S.E.M. Statistical significance was evaluated by using a one-way ANOVA.
Effects of prolonged exposure to alanine on cellular integrity, insulin secretory responses and cellular insulin content
Analysis of cellular integrity, by Neutral Red and MTT assays, following culture of cells in the absence or presence of 10 mmol/l L-alanine revealed that alanine culture did not significantly alter cellular integrity compared with standard culture (Neutral Red assay, 100.0±10.3% for control and 99.7±8.1% for alanine culture; MTT assay, 100.0±17.7% for control and 104.5±4.1% for alanine culture). The insulin-secretory responses to L-alanine after 18 h in culture in the absence or presence of 10 mmol/l L-alanine are shown in Figure 1(A). Although prior exposure to 10 mmol/l L-alanine did not significantly alter basal insulin release at 1.1 mmol/l D-glucose, alanine culture markedly suppressed the subsequent stimulatory effect of acute addition of alanine compared with control cells (74% reduction; P<0.001). This alanine-induced demise in insulin output was partially restored (P<0.01) after a further 18 h recovery period involving culture in the absence of alanine. As shown in Figure 1(B), 18 h exposure to 10 mmol/l L-alanine in culture did not significantly affect insulin content compared with control cells exposed to standard culture medium, and there was evidence of enhanced content after an 18 h recovery period. Taken together, these results indicate that alanine induced β-cell desensitization.
It was also of interest to assess whether the observed desensitization was specific to β-cell alanine responsiveness by assessing the impact of prolonged exposure to alanine on subsequent responses to a depolarizing concentration of KCl or other key nutrient secretagogues (Figure 2). Alanine culture did not alter the marked 6.7-fold (P<0.001) acute insulinotropic action of 30 mmol/l KCl (Figure 2A). Similarly, the insulin-releasing effects of acute stimulation with 16.7 mmol/l D-glucose, 10 mmol/l mannose or 30 mmol/l KIC (2-oxoisocaproic acid) were not affected by alanine culture (Figure 2B). To probe the alanine-induced desensitization mechanism further, cells were cultured with the non-metabolizable amino acid analogue AIB (aminoisobutyric acid), which shares the same transport mechanism as alanine. As with alanine culture, prolonged exposure to AIB in culture did not alter basal insulin release at 1.1 mmol/l D-glucose (Figure 3). Interestingly, alanine culture also served to significantly reduce the subsequent responsiveness to AIB (by 44%; P<0.05), although not to the same extent as the reduction in alanine-induced insulin output (Figure 3). Likewise, AIB culture served to decrease alanine-induced insulin release (by 71%; P<0.001) and the insulinotropic response to AIB (by 63%; P<0.01; Figure 3). Alanine or AIB culture also reduced serine-induced insulin secretion compared with standard culture (P<0.01; Figure 3).
Effects of prolonged exposure to alanine on membrane potential and [Ca2+]i
The impact of culture with 10 mmol/l L-alanine on subsequent membrane potential and [Ca2+]i responses to acute stimulation with alanine (10 mmol/l) is shown in Figure 4. After standard culture in the absence of alanine, BRIN-BD11 cells had a marked membrane depolarization, but AUC (area under the curve) analysis revealed that this increase in membrane potential was significantly (P<0.01) suppressed in cells exposed to alanine culture (Figure 4A), although membrane potential at 1.1 mmol/l D-glucose was not altered (results not shown). The suppressed depolarization response to acute stimulation with 10 mmol/l L-alanine was partly reversed after an 18 h recovery period, consistent with a partial restoration of insulin release.
Corresponding to the membrane-depolarizing effect of alanine after standard culture, acute stimulation with 10 mmol/l L-alanine caused a notable increase in [Ca2+]i (Figure 4B), consistent with the insulin-releasing actions of this amino acid. However, AUC analysis also demonstrated a significant (P<0.01) reduction in the 10 mmol/l L-alanine-induced increase in [Ca2+]i following alanine culture (Figure 4B). At 1.1 mmol/l D-glucose, [Ca2+]i levels were similar following culture in the absence or presence of alanine (results not shown). Again, the reduced increase in [Ca2+]i following alanine culture was partially restored by 18 h recovery in culture medium in the absence of alanine, consistent with the view that the alanine-induced β-cell suppression was a reversible desensitization.
Effects of prolonged exposure to alanine on subsequent alanine metabolism
Following incubation with L-[3-13C]alanine, the end products included glutamate labelled at positions C2, C3 and C4, and aspartate labelled at positions C2 and C3 (Figure 5). The glutamate C4 peak consisted predominantly of a singlet peak due to enrichment at the C4 position only (Figure 5A). A small doublet peak was also present, which was due to glutamate labelled at positions C3 and C4 (Figure 5A). Following 18 h of alanine culture, there was a significant increase in glutamate labelled at position C4, but not at positions C2 and C3 (Figures 5A and 5B). Closer examination of the C4 peak revealed that the C4S (C4 singlet) increased significantly from 18.2±1.9 to 29.6±1.7 nmol/mg of protein (P<0.005). C4S can only be labelled by entry via PDH, indicating increased flux through PDH following prolonged alanine exposure .
Calculation of the specific enrichment showed that there was no change for glutamate positions C2 and C3 (Figure 5C); however, the percentage enrichment increased significantly in position C4 (Figure 5C). The amount of 13C-labelled alanine in the extracts did not significantly change following prolonged exposure to alanine (0.53±0.01 and 0.49±0.04 μmol/mg of protein for control and alanine culture respectively). Interestingly, lactate release did not change significantly from control conditions during the 1 h incubation (25.7±1.8 and 22.5±3.6 nmol/mg of protein for control and alanine culture respectively). Furthermore, the amount of L-alanine remaining in the medium at the end of the 1 h incubation was not significantly different following prolonged culture with L-alanine (17.1±3.1 and 11.7±4.0 μmol/mg of protein for control and alanine culture respectively).
Intracellular ATP levels were measured in parallel experiments at the end of the 1 h incubation and, under control conditions, the ATP concentration was 5.8 pmol/104 cells. Alanine culture significantly (P<0.05) reduced the ATP concentration by 28% to 4.2 pmol/104 cells.
Effects of prolonged exposure to alanine on PDK-2 and PDK-4 gene and protein expression
Exposure of BRIN-BD11 cells to alanine in culture for 18 h resulted in a reduction in PDK-2 protein expression, as evaluated by Western blot analysis (Figures 6A and 6B). Consistent with this alteration in protein expression, semi-quantitative RT (reverse transcription)–PCR of PDK-2 gene expression was also suppressed by alanine culture (Figure 6C). Similarly, as shown in Figure 7(A), alanine culture resulted in reduced PDK-4 protein expression and PDK-4 mRNA levels (Figure 7B). These observations are consistent with the altered flux through PDH observed in the NMR experiments.
Prolonged exposure of β-cells to glucose, fatty acids and insulinotropic drugs has been shown to induce β-cell desensitization to subsequent acute stimulation [1,15,21]. More recently, the effects of prolonged (24 h) exposure to alanine or glutamine on gene expression has been reported, revealing an alteration of 66 or 166 genes respectively, with a large functional core related to signalling and metabolism [23,37]. These studies also indicated that prolonged alanine exposure may confer protection against cytokine-induced β-cell apoptosis. However, although the detrimental actions of glucose and lipids on β-cell function are well-characterized, the consequences of prolonged amino acid exposure on β-cell metabolism, insulin secretion and function has been largely neglected, prompting the present studies. The importance of the present study is emphasized by the fact that alanine is quantitatively the second most abundant amino acid in blood and extracellular tissues in vivo.
Following 18 h of culture with alanine, there was a marked impairment in subsequent characteristic potent insulin-releasing actions of this amino acid. Alanine culture did not, however, significantly diminish basal insulin release or cellular insulin content. Furthermore, when cells exposed to alanine for 18 h were cultured for a further 18 h in the absence of this amino acid, there was a partial restoration of alanine-induced insulin release. This would indicate a reversible desensitization, as reported previously with other insulinotropic agents [15,38,39]. Consistent with a specific alanine-induced amino acid desensitization, responses to depolarizing concentrations of KCl, stimulatory glucose, the insulinotropic hexose sugar mannose or KIC were not affected by chronic alanine culture.
The mode of action by which alanine stimulates insulin release involves both co-transport into cells with Na+ and the rapid intracellular metabolism to generate ATP and other metabolic signalling factors [12–14,40,41]. The insulinotropic response to a non-metabolizable amino acid analogue of alanine, AIB, was significantly reduced following alanine culture albeit to a lesser extent than alanine-induced insulin output. Similarly, AIB culture significantly impaired both alanine- and AIB-induced insulin release, which would further indicate desensitization of the amino acid/Na+ co-transport system. However, it should be noted that the acute uptake of alanine did not change following prolonged culture with alanine, as indicated by the amount of 13C-labelled alanine in the extracts. In the present study, alanine induced a rapid membrane depolarization, reaching a maximum peak at approx. 30 s, and this response was markedly decreased in cells cultured for 18 h with alanine. The depolarizing action of alanine is consistent with acute effects of this and other amino acids co-transported with Na+ . Such amino acids evoke membrane depolarization and increase Ca2+ influx, whereas metabolizable amino acids (including alanine) will additionally increase intracellular ATP concentrations and other signalling factors, prompting KATP channel closure and insulin exocytosis . Indeed, the initial rapid increase in membrane potential observed in the present study indicates potent membrane depolarizing actions, largely attributable to Na+ co-transport and accumulation, whereas the sustained plateau probably reflects the actions of cellular metabolic signalling factors, including ATP acting through KATP channels and other signal elements, modulating [Ca2+]i and insulin release .
Consistent with other studies, acute exposure to alanine resulted in a rapid increase in intracellular Ca2+ concentration [34,42]; however, in cells cultured with alanine, the maximal [Ca2+]i peak was markedly reduced, correlating with suppressed membrane depolarization, an effect which was partially reversed following an 18 h recovery period, again suggesting non-permanent alanine-induced desensitization with prolonged exposure. Although the increase in [Ca2+]i reflects co-transport with Na+ and metabolic ATP generation, the decreased response following prolonged alanine exposure prompted additional studies investigating L-alanine metabolism and ATP generation in cells cultured with L-alanine.
To explore the relevance of metabolic changes to the diminished insulin-secretory responses to alanine following prolonged alanine exposure, metabolism was traced using 13C NMR analyses. We have previously utilized this approach to characterize nutrient metabolism, taking advantage of cultured pancreatic BRIN-BD11 β-cells to enhance the understanding of complex metabolic pathways [12,25,43,44]. The present study revealed a significant increase in flux through PDH following 18 h of alanine culture, as indicated by an increase in the C4S peak of glutamate. Interestingly, this increased flux through PDH should subsequently lead to an increase in NADH concentrations and subsequently enhancement of ATP production; however, alanine culture resulted in a small, but significant, decrease in ATP concentration, despite a notable increase in oxidative metabolism. Nevertheless, as ATP is an important modulator of the depolarizing and Ca2+-dependent insulin-releasing actions of alanine, this observation is consistent with findings arising from other components of the present study. It should be noted that the changes in ATP levels did not affect the viability of the cells.
The activity of the β-cell Na+/K+ pump has been reported to consume 75–80% of basal energy production in these cells , and thus it is possible that the decrease in cellular ATP may be related to enhanced Na+/K+ pump activity following prolonged alanine exposure. A study by Elmi and co-workers  investigated the relationship between islet ATP content and pump activity, demonstrating that pump inhibition with ouabain did not adversely affect ATP levels during acute incubations. However, upon longer incubation, a very different pattern emerged, with decreased islet ATP concentration corresponding to reduced glucose oxidation, opposing the expected outcome of inhibition of the ATP-consuming Na+/K+ pump. Moreover, diazoxide has been demonstrated to reduce the basal activity of the pump and elevate islet ATP content , but the opposite has also been reported , making it difficult to draw clear conclusions on the relationship between ATP concentrations and the activity of the Na+/K+ pump. Considering that alanine is present in both our control and pre-cultured conditions and that acute alanine uptake did not change significantly, it is unlikely that the reduction in ATP at the end of the 1 h incubation is due to increased activity of the Na+/K+ pump. However, it is possible that the observed alanine-induced decrease in ATP despite increased oxidative metabolism could result from an uncoupling of ATP production in mitochondria resulting from ROS (reactive oxygen species) generated as a by-product of oxidative metabolism.
Given the observation that prolonged alanine exposure resulted in an increased flux through PDH, it was of interest to examine the expression of the major inhibitory PDKs (PDK-2 and PDK-4). It should be noted that the regulation of PDH activity is complex and that the PDKs represent only part of the regulation of PDH activity . PDKs control PDH activity through phosphorylation, causing reduced enzyme activity . Notably, several studies have linked altered PDH activity with a decline in β-cell function, and expression of PDK-1, PDK-2 and PDK-4 has been reported in pancreatic β-cells [50,51]. Phosphorylation of PDH occurs at three specific sites and PDK-2 has the highest activity at site 1, whereas PDK-4 has the highest activity towards site 2 . The present findings demonstrating the down-regulation of PDK-2 and PDK-4 mRNA levels following alanine culture, using semi-quantitative RT–PCR, are entirely consistent with the observed increased flux through PDH. Prolonged alanine culture also down-regulated PDK-2 and PDK-4 protein expression, again being consistent with an increased flux through PDH. Interestingly, a similar down-regulation of PDK-4 was reported following exposure to high glucose , and up-regulation of PDK-4 has been reported to lower PDH activity during starvation , allowing the entry of acetyl-CoA derived from fatty acids.
Collectively, the results of the present study demonstrate the alanine-induced down-regulation of important PDK isoforms in β-cells, resulting in the observed increase in PDH activity/flux, which has the knock-on effect of reducing tricarboxylic acid cycle entry via PC (pyruvate carboxylase) and reduced entry of acetyl-CoA from fatty acid oxidation. Consequently, as PC flux is important for fuel-driven insulin secretion [52–54], the decreased flux through PC may at least partly account for the reduction in insulin secretion following prolonged alanine exposure. Further studies, including a time course of the response, are required to explore these possibilities, to test whether the desensitization observed in the present study with alanine and related AIB extends to other functionally important amino acids, such as leucine and glutamine, and to evaluate the role of ROS and other metabolic intermediates in amino-acid-induced β-cell desensitization. Notably, however, the results of the present study clearly demonstrate that prolonged exposure to alanine can induce β-cell desenstitization, which has an impact upon cellular metabolism and the amino acid regulation of cellular metabolic and ionic flux.
This work was supported by the Health Research Board and by UCD (to L. B.); and by the Research and Development Office of the Northern Ireland Health and Personal Social Services (NI HPSS).
Abbreviations: AIB, aminoisobutyric acid; AUC, area under the curve; [Ca2+]i, intracellular [Ca2+]; C4S, C4 singlet; FBS, foetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; KATP channel, ATP-sensitive K+ channel; KIC, 2-oxoisocaproic acid; KRBB, Krebs–Ringer bicarbonate buffer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PDK, PDH kinase; ROS, reactive oxygen species; RT–PCR, reverse transcriptase–PCR
- © The Authors Journal compilation © 2009 Biochemical Society