Chronic exposure to leucine in vitro induces β-cell dysfunction in INS-1E cells and mouse islets

in Journal of Endocrinology

Chronic hyperglycemia and hyperlipidemia cause deleterious effects on β-cell function. Interestingly, increased circulating amino acid (AA) levels are also a characteristic of the prediabetic and diabetic state. The chronic effects of AAs on β-cell function remain to be determined. Isolated mouse islets and INS-1E cells were incubated with or without excess leucine. After 72 h, leucine increased basal insulin secretion and impaired glucose-stimulated insulin secretion in both mouse islets and INS-1E cells, corroborating the existence of aminoacidotoxicity-induced β-cell dysfunction. This took place concomitantly with alterations in proteins and genes involved in insulin granule transport, trafficking (e.g. collapsin response mediator protein 2 and GTP-binding nuclear protein Ran), insulin signal transduction (proteasome subunit α type 6), and the oxidative phosphorylation pathway (cytochrome c oxidase). Leucine downregulated insulin 1 gene expression but upregulated pancreas duodenum homeobox 1 and insulin 2 mRNA expressions. Importantly, cholesterol (CH) accumulated in INS-1E cells concomitantly with upregulation of enzymes involved in CH biosynthesis (e.g. 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate (diphospho) decarboxylase, and squalene epoxidase) and LDL receptor, whereas triglyceride content was decreased. Our findings indicate that chronic exposure to elevated levels of leucine may have detrimental effects on both β-cell function and insulin sensitivity. Aminoacidotoxicity may play a pathogenic role in the development of type 2 diabetes.

Abstract

Chronic hyperglycemia and hyperlipidemia cause deleterious effects on β-cell function. Interestingly, increased circulating amino acid (AA) levels are also a characteristic of the prediabetic and diabetic state. The chronic effects of AAs on β-cell function remain to be determined. Isolated mouse islets and INS-1E cells were incubated with or without excess leucine. After 72 h, leucine increased basal insulin secretion and impaired glucose-stimulated insulin secretion in both mouse islets and INS-1E cells, corroborating the existence of aminoacidotoxicity-induced β-cell dysfunction. This took place concomitantly with alterations in proteins and genes involved in insulin granule transport, trafficking (e.g. collapsin response mediator protein 2 and GTP-binding nuclear protein Ran), insulin signal transduction (proteasome subunit α type 6), and the oxidative phosphorylation pathway (cytochrome c oxidase). Leucine downregulated insulin 1 gene expression but upregulated pancreas duodenum homeobox 1 and insulin 2 mRNA expressions. Importantly, cholesterol (CH) accumulated in INS-1E cells concomitantly with upregulation of enzymes involved in CH biosynthesis (e.g. 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate (diphospho) decarboxylase, and squalene epoxidase) and LDL receptor, whereas triglyceride content was decreased. Our findings indicate that chronic exposure to elevated levels of leucine may have detrimental effects on both β-cell function and insulin sensitivity. Aminoacidotoxicity may play a pathogenic role in the development of type 2 diabetes.

Keywords:

Introduction

Type 2 diabetes (T2D) involves both defective insulin secretion and insulin resistance (IR). In T2D, hyperglycemia and hyperlipidemia exert deleterious effects on β-cell function, referred to as glucotoxicity and lipotoxicity (Xiao et al. 2001, El-Assaad et al. 2010), with increased basal insulin secretion (BIS) and impaired glucose-stimulated insulin secretion (GSIS). Patients with impaired glucose tolerance (IGT) or in the early stage of T2D present with characteristic β-cell dysfunction (Ferrannini et al. 2003), but the nature of the primary β-cell defect is still elusive.

In an IR state, plasma concentrations of amino acids (AAs) are elevated, particularly leucine, isoleucine, valine, proline, and glutamine (Newgard et al. 2009). Thus, elevated levels of certain AAs are also a characteristic of the diabetic state and may play a pathogenic role in the development of T2D in addition to hyperglycemia and dyslipidemia.

Most AAs, including leucine, acutely stimulate insulin secretion from pancreatic β cells in a dose- and glucose-dependent manner (Liu et al. 2008), and leucine stimulates insulin secretion by serving as both a metabolic fuel and an allosteric activator of glutamate dehydrogenase (Newsholme et al. 2006, MacDonald et al. 2008). Leucine and its transaminated product α-ketoisocaproate may also affect insulin secretion via direct inhibition of β-cell ATP-regulated potassium channel currents (Newsholme et al. 2006). Correspondingly, acute in vivo studies consistently revealed that additional leucine intake increased insulin secretion and reduced postprandial blood glucose (Kalogeropoulou et al. 2008). However, there are conflicting results on the long-term effects of leucine on β-cell function. Long-term exposure of pancreatic islets to high leucine levels has been shown to selectively impair GSIS, which is associated with a reduced ATP-to-ADP ratio (Anello et al. 2001). Moreover, Zhang et al. (2009) reported that chronic exposure to leucine downregulates the expression of pancreas duodenum homeobox 1 (Pdx1), glucokinase (Gk (Gck)), and glucose transporter type 2 in rat insulinoma β cells, resulting in decreased insulin content and GSIS at high glucose. However, this was challenged by the observation that leucine upregulates the ATP synthase β subunit (ATPβ) mRNA level in RIN5mf cells and increases GSIS, ATP, GCK, and ATPβ levels in rat and human islets (Yang et al. 2006). Conflicting results were also observed when rats were fed leucine orally for varying number of weeks. Mice given a high-fat diet with doubled dietary leucine intake were significantly more glucose tolerant and insulin sensitive than high-fat diet mice without additional leucine (Zhang et al. 2007). By contrast, Balage et al. (2011) observed that leucine-supplemented rats displayed IGT and had a significant increase in visceral adipose tissue.

Given such conflicting results on the actions of leucine, we wanted to elucidate the long-term actions of leucine on β-cell function in vitro. Here, we show that chronic exposure to excess leucine induces characteristic diabetic changes in β-cell function (i.e. increases BIS and hampers GSIS) and alters β-cell gene and protein expressions. We found that leucine-induced β-cell dysfunction is related to abnormal cholesterol (CH) metabolism with corresponding accumulation of CH content, impaired oxidative phosphorylation (OxPhos), impaired insulin secretory granules (ISGs) transport, and trafficking.

Materials and Methods

Reagents and buffers/solutions

All chemicals were from Sigma–Aldrich if not stated otherwise. Modified Krebs–Ringer buffer (M-KRB) contained (mM) NaCl, 125; KCl, 5.9; MgCl2, 1.2; CaCl2, 1.28; NaHCO3, 5.0; and HEPES, 25; pH 7.4. SYTO 24 solution: 5 mM SYTO 24 green fluorescent nucleic acid stain (Molecular Probes, Invitrogen, Eugene, OR, USA) dissolved in DMSO (≥99.9%) to a final concentration of 0.01 mM. Glycine–BSA buffer (pH 8.8) contained glycine 100 mM and 0.25% BSA. 2D-lysis buffer contained 6 M urea, 2 M thiourea, 1.5% (w/v) pharmalyte, 0.8% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, and 1% (w/v) dithioerythritol in water. The rehydration buffer consisted of the same substances, in the same concentrations as the lysis buffer, but with extra pharmalyte (5 μl/ml).

Animals

Adult female NMRI mice (Bomholtgaard Breeding and Research Centre, Ry, Denmark) weighing 20–25 g were used. The animals were kept on a standard pellet diet and tap water ad libitum and a 12 h light:12 h darkness cycle. The study was carried out in accordance with the guidelines of the Danish Council for Animal Experiments.

Islet isolation

Islets were isolated by the collagenase digestion technique (Lacy & Kostianovsky 1967). In brief, the animals were anesthetized i.p. with pentobarbital sodium (50 mg/kg), and a midline laparotomy was performed. The pancreas was retrogradely filled with 3 ml ice-cold Hanks Balanced Salt Solution (HBSS) supplemented with 0.3 mg/ml collagenase P (Boehringer Mannheim). The pancreas was removed, incubated for 19 min at 37 °C in a water bath, and subsequently rinsed with ice-cold HBSS, after which the islets were handpicked under a stereomicroscope. The islets were incubated overnight at 37 °C and 95% normal atmosphere-5% CO2 in 10 ml RPMI 1640 containing 11.1 mM glucose supplemented with 10% FBS, 2.06 mM glutamine, 100 IU/ml penicillin G, and 100 μg/ml streptomycin (all Gibco-BRL).

Insulin secretion from islets

After overnight incubation, mouse islets were incubated in RPMI 1640 with 11.1 mM glucose in the presence of 1, 5, and 10 mM leucine, respectively, the normal RPMI 1640 containing 0.38 mM leucine as control (all the following experiments take 0.38 mM leucine as control). After 72-h leucine treatment, islets were rinsed once with M-KRB supplemented with 3.3 mM glucose and 0.1% human serum albumin and preincubated for 30 min in M-KRB at 37 °C. Consequently, a single islet was handpicked and incubated in 100 μl M-KRB with 3.3 or 16.7 mM glucose. After 60 min of incubation in normal atmosphere at 37 °C, the medium (50 μl) was collected and frozen for analysis of insulin.

Cell culture

INS-1E cells (generous gift from Prof. Claes B Wollheim, Geneva, Switzerland) were cultured in RPMI 1640 medium containing 11.1 mM d-glucose and supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 2 mM l-glutamine, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol, at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. The cells were passaged weekly.

Insulin secretion from INS-1E cells

The INS-1E cells were seeded (3.0×105 cells/well) onto 24-well Black Visiplate TC (Wallac Oy, Turku, Finland) plates in 1 ml RPMI 1640. After adhering overnight, the cells were cultured in RPMI 1640 with 11.1 mM glucose in the presence of 0.38, 1, 5, and 10 mM leucine for 72 h in a humidified atmosphere (5% CO2 95% air at 37 °C). Afterward, the cells were preincubated with M-KRB supplemented with 3.3 mM glucose and 0.1% human serum albumin for 30 min and then the cells were incubated in 1 ml M-KRB containing 3.3 or 16.7 mM glucose for 1 h. Subsequently, supernatants (300 μl) were collected, centrifuged, and 200 μl were kept at −20 °C for insulin analysis. After the secretion study, the cells were washed once with 1 ml PBS (Gibco), and the number of cells was estimated using nuclear staining with SYTO 24 reagent (20 μl/well) and measured by FLUOstar Galaxy (BMG, Ramcon, Denmark). Unless otherwise stated, passage numbers between 60 and 82 were used.

Insulin output of INS-1E cells

INS-1E cells were incubated and treated as earlier. For the insulin output study, incubation medium (25 μl) was sampled after 24, 48, and 72 h and stored at −20 °C until insulin analysis.

Insulin content of INS-1E cells

The INS-1E cells were incubated and treated as earlier, but in six-well plates with a density of 1.0×106 cells/well in 3 ml RPMI 1640. After 72 h, the cells were washed once with 2 ml cold PBS and then 1 ml glycine–BSA buffer was added. Cells were scratched and sonicated twice at 0 °C for 14 s (Branson Sonifier 250, Danbury, CT, USA). We took 250 μl to evaluate the total protein with a detergent-compatible protein kit (Bio-Rad Laboratories) for calibration of insulin content. The remaining 750 μl was centrifuged for 30 min at 21460 g and the supernatant was collected and frozen at −20 °C for a later insulin assay.

Insulin assay

Insulin was analyzed by RIA using a guinea pig antiporcine insulin antibody (Novo Nordisk, Bagsvaerd, Denmark) and mono-125I-(Tyr A14)-labeled human insulin (Novo Nordisk A/S) as tracer and rat insulin as standard (Novo Nordisk A/S). The separation of bound and free radioactivity was performed using ethanol. The intra- and interassay coefficients of variation were determined to be <10%.

Determination of triglyceride and CH content

INS-1E cells were plated at 3.0×105 cells/well in 1 ml of medium in 24-well Black Visiplate TC plates. The cells were allowed to adhere overnight and then incubated and treated as earlier. After 72 h, the cells were washed once with 1 ml PBS, and the number of cells was estimated as earlier. The medium was removed, and the cells were frozen for 1 h at −80 °C. Subsequently, the cells were treated for 20 min with triglyceride (TG) reagents (250 μl/well, Roche) at room temperature. TG content was determined with a TG GPO-PAP kit (Roche) and normalized to cell number. The recovery of TG was ∼90%.

Correspondingly, cells were treated for 20 min with CH reagents (250 μl/well, Roche) at room temperature. CH content was determined by a CH CHOD-PAP kit (Roche) and was normalized to cell number. The recovery of CH content was ∼90%.

3H-thymidine incorporation in INS-1E cells

3H-thymidine incorporation was used to monitor INS-1E cell proliferation and DNA synthesis. Briefly, INS-1E cells (4×104/well) were seeded in 96-well isoplates (Wallac Oy) and cultured in RPMI 1640 with 11.1 mM glucose and 10% FBS. On the next day, the cells were incubated with ‘starvation medium’ (RPMI 1640 with 0.5 mM glucose, 0.1% BSA, without FBS) for 24 h. The cells were then cultured for another 72 h in RPMI 1640 containing 0.5% BSA, 11.1 mM glucose in the presence of 0.38, 1, 5, and 10 mM leucine, and 1 μCi [methyl-3H]thymidine (Perkin Elmer, Wellesley, MA, USA). After 72 h, the cells were washed twice in cold PBS and 200 μl liquid scintillator was applied. The incorporated [methyl-3H]thymidine was counted by a 1450 MicroBeta TRILUX (Wallac).

The incidence of dead INS-1E cells

INS-1E cells were seeded in 96-well Black Visiplate TC plates (Wallac Oy) at a density of 4×104 cells/well in 200 μl medium, the cells were allowed to adhere overnight, and then treated and cultured with different concentrations of leucine as earlier. After 72 h, the number of dead cells in each well was calculated using a fluorometric assay kit based on the cell lysis and staining method (Sakai et al. 2001; Cytotoxic Fluoro-test Wako; Wako Pure Chemical Industries, Osaka, Japan) in the FLUOstar Galaxy. A linear relationship was confirmed in advance between the viable cell density and fluorescence intensity.

RNA extraction and cDNA synthesis

Three different passages of INS-1E cells were incubated in RPMI 1640 in the presence of 0.38 or 5 mM leucine for 72 h. The RNA of treated cells was extracted using the AllPrep RNA/Protein kit (Qiagen) according to the manufacturer's instructions (AllPrep RNA/Protein Handbook; Qiagen) on the QIAcube machine (Qiagen). RNA concentration and purity were determined by measuring the absorbance at 260 and 280 nm (NanoDrop ND-8000 u.v.–Vis Spectrophotometer, NanoDrop Technologies Wilmington, DE, USA). The RNA quality was evaluated by examining the 18s and 28s ribosomal band on a 1% nondenaturing agarose gel, stained with SYBR green.

RT was performed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. We applied 1 μg total RNA for each 20 μl RT reaction.

Quantitative RT-PCR

RT-PCR was performed using an Applied Biosystems 7500 FAST PCR machine (Applied Biosystems). Predesigned TaqMan probes and primers with the following catalog numbers were obtained from Applied Biosystems: Pdx1 (Rn00755591_m1), the v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA, Rn00845206_s1), insulin 1 (Ins1, Rn02121433_g1), insulin 2 (Ins2, Rn01774648_g1), acetyl-CoA carboxylase α (Acc1 (Acaca), Rn00573474_m1), fatty acid synthase (Fasn, Rn01463548_m1), sterol-regulatory element binding factor 2 (Srebf2, Rn01306296_m1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr, Rn00565598_m1), mevalonate (diphospho) decarboxylase (Mvd, Rn00579216_m1), squalene epoxidase (Sqle, Rn00567532_m1), LDL receptor (Ldlr, Rn00598442_m1), cytochrome c oxidase (Cox), subunit VIa, polypeptide 2 (Cox6a2, Rn00563091_g1), caspase-3 (Casp3, Rn00563902_m1), death-associated protein kinase 3 (Dapk3, Rn00574826_m1), Bcl2 modifying factor (Bmf, Rn00594968_m1), and eukaryotic 18S (Hs99999901_s1).

The RT-PCR mix contained 5 μl of 2×TaqMan FAST Universal Master Mix (P/N 43660783, Applied Biosystems), 0.5 μl of 20×TaqMan Assay/probe (Applied Biosystems), and cDNA equivalent to 50 ng total RNA in 4.5 μl H2O. The thermal FAST cycle program was one cycle at 95 °C for 20 s, 40 cycles at 95 °C for 3 s, and 60 °C for 30 s. Each PCR sample was performed in six repeats, and gene expressions were normalized to eukaryotic 18S expression. All assays were carried out in 96-well plates with an optical adhesive cover (P/N 4346906 and P/N 4311971, Applied Biosystems). We used the 2ΔΔCT method to calculate the relative gene expression (Pfaffl 2001). No template controls and no amplification controls were included for each gene as negative controls.

INS-1E cell growth and lysis for proteomics

Protein from three different passages of INS-1E cells was extracted at the same time as RNA was purified on the QIAcube machine. We took two samples every time for each condition; as a result, there were six replicates of each treatment used for proteomics. The protein content of the cell suspensions was analyzed by the Bradford assay (Bio-Rad). In order to obtain sufficient protein concentration of the cell suspensions, the samples were precipitated with addition of 250 μl 24% trichloroacetic acid at 4 °C and left to precipitate for 30 min at 0 °C, followed by centrifugation at 6000 g at 0 °C for 10 min. The supernatant was removed, the protein pellet was washed twice by addition of 500 μl acetone at 0 °C, and centrifuged as described between each washing step. The protein pellets were kept at −80 °C for proteomic analysis.

Proteomic analyses

The 2-dimensional gel electrophoresis (2-DGE) analysis was performed essentially as described earlier (Young et al. 2010). Briefly, the stored protein pellets were thawed and solubilized in 250 μl of 2D lysis buffer and subsequently tenfold in Millipore water. Based on protein determination using the Bradford assay, a sample volume corresponding to 100 μg protein was applied to each gel. The 12 lysed cell samples were analyzed in a single 2-DGE gel set consisting of six gels representing either control INS-1E cells or treated with leucine, respectively, as described earlier using 11 cm IPG strips (pH 5–8) for the first dimension and 12.5% Criterion gels (Bio-Rad) for the second dimension. Analytical gels were stained with Flamingo Pink (Bio-Rad) and scanned using a Molecular Imager FX (Bio-Rad). Images were analyzed with PDQuest (Bio-Rad). Gels for spot identification were stained with colloid Coomassie Blue R-250 (Kang et al. 2002). In-gel digestion for peptide mass fingerprinting, desalting, and concentration of protein spots, identification of MFGM proteins by matrix-assisted laser desorption ionization–time of flight mass spectrometry, and image analysis was performed as described earlier (Larsen et al. 2010).

Statistical analysis

Data are presented as mean±s.e.m. We compared groups using one-way ANOVA; we also performed statistical analysis using the unpaired two-tailed Student's t-test with unequal variances. Each treatment condition was compared with controls with Bonferroni's correction. Differences of P<0.05 were considered to be significant. All statistical analysis was performed with GraphPad Prism Software (San Diego, CA, USA).

Results

Impact of leucine on insulin secretion from isolated islets and INS-1E cells

Figure 1A shows that at 3.3 mM glucose BIS from isolated islets increased after 72-h incubation with 1, 5, or 10 mM leucine. By contrast, glucose (16.7 mM)-stimulated insulin secretion decreased after 72-h exposure of the islets to 1, 5, or 10 mM leucine (Fig. 1A). Similar results were obtained in INS-1E cells, demonstrating increased BIS and decreased GSIS after 72-h exposure to high leucine concentrations (Fig. 1B). We did not find any change in insulin content after 72-h exposure to 1, 5, and 10 mM leucine compared with control (0.928±0.129 ng/μg protein (control), 0.812±0.136 ng/μg protein (1 mM leucine), 0.832±0.107 ng/μg protein (5 mM), 0.884±0.131 ng/μg protein (10 mM)); after 72 h, no change in insulin output was detected with 1, 5, and 10 mM leucine (386.7±10.5 ng/ml per 10 000 cells (control), 388.8±16.4 ng/ml per 10 000 cells (1 mM leucine), 365.2±7.2 ng/ml per 10 000 cells (5 mM), and 392.4±11.0 ng/ml per 10 000 cells (10 mM)).

Figure 1
Figure 1

(A) Effects of 72-h incubation with leucine on the response of mouse islets to glucose. The insulin secretion from mouse islets at low (3.3 mM) (ΔP<0.05, ΔΔP<0.01) and high (16.7 mM) glucose concentrations (*P<0.05 and **P<0.01) for 1 h compared with control (0.38 mM leucine). This part includes three independent experiments. For each experiment, we applied the mean value from 8 to 11 islets for each condition. (B) Effects of 72-h incubation with leucine on the response of INS-1E cells to glucose. The insulin secretion from per 10 000 INS-1E cells at low (3.3 mM) (ΔP<0.05, ΔΔP<0.01) and high (16.7 mM) glucose concentrations (**P<0.01, ***P<0.001) for 1 h compared with control (0.38 mM leucine). This part includes three independent experiments. For each experiment, we applied the mean value from six samples for each condition.

Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0148

Effects of 72-h exposure to leucine on gene expressions in INS-1E cells

The impact of 72-h incubation with 5 mM leucine on gene expression was studied in INS-1E cells for a number of β-cell genes (Fig. 2A, B, C, D, E, F, G, H, I, J, K, and L). Leucine upregulated Pdx1 expression (Fig. 2A), did not change MafA expression (Fig. 2B), downregulated Ins1 expression (Fig. 2C), and upregulated Ins2 expression (Fig. 2D). As shown in Fig. 2E and F, leucine increased Acaca and Fasn expressions in INS-1E cells.

Figure 2
Figure 2

Changes in gene expressions in INS-1E cells induced by 5 mM leucine. The following genes were investigated: Pdx1 (A), MafA (B), Ins1 (C), Ins2 (D), Acaca (E), Fasn (F), Srebf2 (G), Hmgcr (H), Mvd (I), Sqle (J), Ldlr (K), and Cox6a2 (L). *P<0.05, **P<0.01, ***P<0.001 vs control, ΔΔΔP<0.001 vs control. RNA extraction includes three experiments applying different passage number cells. RT-PCR was performed in six repeats.

Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0148

As depicted in Fig. 2G, leucine significantly increased Srebf2 mRNA expression. Figure 2H, I and J demonstrate changes in the expression of a series of genes encoding for enzymes involved in CH biosynthesis in INS-1E cells exposed to leucine. Hmgcr, Mvd, and Sqle were upregulated. Leucine also increased Ldlr mRNA level in INS-1E cells (Fig. 2K). By contrast, leucine pronouncedly reduced Cox6a2 mRNA level in INS-1E cells (Fig. 2L).

Influence of leucine on TG and CH content in INS-1E cells

As seen in Fig. 3A, 72-h incubation with leucine (1, 5, or 10 mM) reduced the TG content of INS-1E cells significantly. Figure 3B demonstrates that the CH content in INS-1E cells was increased after 72-h culture with 1, 5, or 10 mM leucine respectively.

Figure 3
Figure 3

(A) Effect of leucine on TG content of INS-1E cells. Changes in TG content per 10 000 INS-1E cells after 72-h incubation with leucine (1–10 mM). ΔΔΔP<0.001 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from six samples for each condition. (B) Effect of leucine on CH content of INS-1E cells. Changes in CH content per 10 000 INS-1E cells after 72-h incubation with 1–10 mM leucine, *P<0.05, **P<0.01 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from six samples for each condition.

Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0148

Effect of leucine on gene expressions associated with apoptosis in INS-1E cells

Leucine (5 mM) did not affect the mRNA levels of Casp3 (Fig. 4A) and Dapk3 (Fig. 4B) in INS-1E cells, whereas the pro-apoptotic protein Bmf (Fig. 4C) mRNA was downregulated.

Figure 4
Figure 4

Impact of leucine on the expressions of genes associated with apoptosis. Alterations of mRNA expression in genes associated with apoptosis in INS-1E cells induced by 72 h exposure to 5 mM leucine. Casp3 (A), Dapk3 (B), Bmf (C), ΔΔΔP<0.001 vs control. RNA extraction includes three independent experiments. RT-PCR was performed in six repeats.

Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0148

Impact of leucine on the viability of INS-1E cells and 3H-thymidine incorporation in INS-1E cells culture

While the percentage of dead INS-1E cells was unchanged by 1 and 5 mM leucine, 10 mM leucine augmented the number of dead INS-1E cells (Fig. 5A). Leucine (1, 5, and 10 mM) decreased 3H-thymidine incorporation in INS-1E cells, indicating a leucine-induced suppression of INS-1E cell proliferation (Fig. 5B).

Figure 5
Figure 5

(A) Impact of leucine on the viability of INS-1E cells. Impact of 72-h incubation with leucine (1–10 mM) on the viability of INS-1E cells, ***P<0.001 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from ten samples for each condition. (B) Effect of leucine on the incorporation of 3H-thymidine into INS-1E cells. Effect of 72-h incubation with leucine (1–10 mM) on 3H-thymidine incorporation in INS-1E cells, ΔΔP<0.01, ΔΔΔP<0.001 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from ten samples for each condition.

Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0148

Proteomics studies: alteration in proteins in INS-1E cells induced by leucine

Results from proteomic studies are presented in Table 1. Leucine (5 mM) increased the autophagy-related enzyme, cathepsin D. Leucine decreased serine/threonine-protein phosphatase PP1, which is related to phosphorylation/dephosphorylation states of proteins. Leucine decreased GTP-binding nuclear protein and increased dihydropyrimidinase-related protein 2 (DRP2), which are related to ISG transport/trafficking. Proteasome subunit α type 6 was identified in two spots, one was upregulated, while the other was downregulated; the two spots are probably active and inactive forms of proteasome subunit α type 6.

Table 1

INS-1E cellular proteins identified by MS and significantly affected by leucine. Fold change (FC) in mean relative spot volumes was calculated relative to the control, FC >1 indicates upregulation, FC <1 indicates downregulation

Protein nameSwiss Prot Acc. no.aMouse scorebt-test (P)Fold change
Pyruvate dehydrogenase E1 component subunit βP4943273*0.021.3
Cathepsin DP24268590.0021.5
Serine/threonine-protein phosphatase PP1P62138115*0.040.9
Dihydro pyridiminase-related protein 2P4794270*0.031.4
Proteasome subunit α type 6P6090189*0.030.8
Proteasome subunit α type 6P6090182*0.00032.1
GTP-binding nuclear proteinP6282865*0.050.5

Primary accession key in the SWISS-PROT database.

Score of the Mascot search. Significant scores (P<0.05) are marked by *.

Discussion

In this study, we demonstrated that long-term exposure to leucine caused β-cell dysfunction with increased BIS and decreased GSIS in both mouse islets and INS-1E cells respectively. The functional derangement of β cells took place concomitantly with alterations in a large number of β-cell gene expressions. For example, Ins1 gene expression was downregulated by chronic exposure to high leucine levels (Fig. 2C), being in line with the observation that GSIS was inhibited and that Ins1 accounts for 60–90% of total insulin immunoreactivity in β-cell lines (Linde et al. 1993). The expressions of the two insulin genes have been proposed to be independently regulated as well as coordinately regulated in both rats and mice (Linde et al. 1993). The differential effect of leucine on expressions of Ins1 and Ins2 mRNA (Fig. 2D) appears puzzling. There was no change in total insulin content or output in INS-1E cells. Pdx1 and MafA are potent stimulators of the transcription of insulin genes (Docherty et al. 2005), and the increased Pdx1 mRNA expression found in this study (Fig. 2A) is very interesting; it is in accordance with increased mRNA expression of Pdx1 in isolated islets from T2D patients, despite less insulin release than control islets in response to glucose (Del Guerra et al. 2005); furthermore, it has previously been shown that glutamine upregulates Pdx1 expression in BRIN-BD11 cells (Corless et al. 2006).

In a global gene expression profiling study (Liu et al. 2011), the largest cluster of genes in INS-1E cells induced by exposure to excess leucine is those regulating CH metabolism. AMP-activated protein kinase (AMPK) was downregulated (Liu et al. 2011), Srebf2 mRNA expression was enhanced (Figs 2G and 6), and as many as ten enzymes in the CH biosynthetic pathway were upregulated (Liu et al. 2011). In line with the change of AMPK in INS-1E cells after prolonged exposure to leucine, islets from T2D patients demonstrated that the percentage of activated AMPK protein was significantly lower than control islets (Del Guerra et al. 2005). The principal targets for phosphorylation by AMPK are Hmgcr and Acaca (Towler & Hardie 2007). Interestingly, Srebf2 gene is tightly associated with the regulation of a number of genes encoding for enzymes involved in the CH biosynthetic pathway (Ishikawa et al. 2008). Our RT-PCR results showed that leucine upregulated expressions of CH biosynthetic enzymes, e.g. acetyl-CoA acetyltransferase 2, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Liu et al. 2011), Hmgcr, Mvd, Sqle (Fig. 2H, I, and J), and Ldlr (Fig. 2K). In line with this, we found that the CH content was significantly increased in INS-1E cells chronically exposed to elevated leucine levels (Fig. 3B). The series of events described here is coherent with several data obtained in vitro and in vivo in animal models and have recently been put into perspective (Peyot et al. 2010, Vergeer et al. 2010, Kruit et al. 2012). Combined deletion of ATP-binding cassette transporter G subfamily 1 (ABCG1) and the ATP-binding cassette transporter subfamily A member 1 (ABCA1) resulted in increased islet CH levels compared with control, β-cell-specific ABCA1 deficiency, and globally deficient ABCG1; notably, islets lacking both ABCA1 and ABCG1 had an even greater reduction in GSIS compared with islets from mice lacking either ABCA1 or ABCG1 (Kruit et al. 2012). Likewise, high-fat induced obese mice were divided into low responders (LDR) and high responders (HDR) according to their body weight (Peyot et al. 2010). There was a 90% and 60% increase in free CH in islets from HDR and LDR respectively; LDR mice islets displayed impaired GSIS, whereas GSIS of HDR mice islets was almost blunted. Moreover, in ABCA1 heterozygotes, the first-phase insulin response is significantly impaired (Vergeer et al. 2010).

Figure 6
Figure 6

Scheme depicting major pathways relating to the possible effects of leucine on pancreatic β cells. AMPK, AMP-activated protein kinase; CH, cholesterol; CRMP-2, collapsin response mediator protein 2; ISGs, insulin secretory granules; OxPhos, oxidative phosphorylation; Srebf2, sterol-regulatory element binding factor 2; TCA, tricarboxylic acid. Dashed line arrow indicates negative effect, solid line arrow indicates positive effect.

Citation: Journal of Endocrinology 215, 1; 10.1530/JOE-12-0148

Consistent with the downregulation of AMPK, long-term exposure to extra leucine results in upregulation of Acaca and Fasn mRNA expressions in INS-1E cells (Fig. 2E and F). Corless et al. (2006) previously also found increased Acaca expression to glutamine. These suggest that leucine may inhibit FA β oxidation and favor CH and FA synthesis. To our surprise, increased levels of leucine resulted in TG reduction in INS-1E cells (Fig. 3A). One explanation might be that leucine strongly induced an increase in CH biosynthesis and is associated with higher consumption of substrate. This may result in a shortage of substrate for TG synthesis and a reduction in TG content.

INS-1E cells demonstrated a robust suppression of Cox6a2 gene expression assessed by both microarray (Liu et al. 2011) and RT-PCR (Fig. 2L) after prolonged exposure to excess leucine (Fig. 6). Cox represents the terminal and rate-limiting enzyme complex of the OxPhos pathway, which is responsible for ATP production in mitochondria. The critical regulatory role of ATP production by OxPhos is underscored by the observation that the Cox activity of islets from the transgenic MKR mouse (a dominant-negative IGF1 receptor mutation specifically in skeletal muscle) was significantly reduced (Lu et al. 2010). As a result, the ATP/ADP ratio was blunted in response to high glucose, and this was followed by a blocking of GSIS, indicating a reduced mitochondrial oxidative capacity (Lu et al. 2010). Accordingly, advanced glycation end products cause impaired GSIS from mice islets and INS-1 cells by inhibiting Cox activity and ATP synthesis (Zhao et al. 2009). This is in line with observations in human diabetes. The expression of a co-regulated subset of OxPhos in individuals with IGT was also downregulated, indicating that downregualtion of a co-regulated subset of OxPhos precedes onset of T2D (Mootha et al. 2003). Taken together, this highlights Cox as an important mechanism by which leucine impairs GSIS in INS-1E cells. Previously, clonal BRIN-BD11 β cells exposed to alanine for 24 h exhibited impaired alanine-induced insulin secretion (Cunningham et al. 2005). Intriguingly, in CH-enriched β cells, the glucose-mediated increase in cellular ATP content was dramatically reduced, and this was related to a decrease in glucose uptake via glucose transporter 2 and an impairment of mitochondrial metabolism (Lee et al. 2011). The relationship between leucine-induced CH accumulation and leucine-induced Cox downregulation should be further investigated (Fig. 6). Cox activity is also regulated by phosphorylation and dephosphorylation. As many as at least ten of the 13 subunits of Cox could be phosphorylated at serine and/or threonine residues (Thompson 2002). As seen from our proteomics results, we found that serine/threonine-protein phosphatase 1 decreased in leucine-treated INS-1E cells (Table 1). Its significance should be further explored deeply.

DRP2 also known as collapsin response mediator protein 2 (CRMP2) plays a role in the maintenance of intracellular Ca2+ homeostasis by direct binding to cytoplasmic loops of N-type Ca2+ channels (Wang et al. 2010). Moreover, CRMP2 directly bound cytoplasmic dynein and prevented dynein-driven microtubule transport in COS-7 cells (Arimura et al. 2009). Leucine significantly increased CRMP2 content in INS-1E cells (Table 1), indicating a potentially detrimental effect on insulin secretion (Fig. 6), which needs to be corroborated in β cells. Furthermore, by the application of proteomics, we found that leucine reduced the expression of GTP-binding nuclear protein Ran (Table 1) in INS-1E cells, which has been reported to be involved in the regulation of microtubule assembly (Dikovskaya et al. 2010). In accordance with our findings, elevated islet CH accumulation due to a lack of β-cell ABCA1 directly impairs depolarization-induced exocytotic fusion of ISGs (Kruit et al. 2011; Fig. 6). Leucine-induced cellular CH accumulation, CRMP2 upregulation, and GTP-binding nuclear protein downregulation are new findings and deserve further evaluation. These findings may have relevance for ISG exocytotic fusion, ISG transport, and trafficking in pancreatic β cells (Fig. 6).

Another protein identified as being differentially regulated by leucine was proteasome subunit α type 6 (Table 1). Interestingly, insulin receptor substrate 1 (IRS1) is selectively regulated by a proteasome degradation pathway activated during prolonged insulin exposure. This is accomplished through regulation of the amount of IRS1, an important receptor-related component in the insulin-signaling cascade (Zhande et al. 2002). Furthermore, leucine diminished insulin receptor expression (Liu et al. 2011) and upregulated ribosomal protein S6 kinase polypeptide 1 mRNA level in INS-1E cells (Liu et al. 2011), the latter inhibited serine phosphorylation of IRS1 to impair insulin action (Tremblay et al. 2005). Consequently, leucine appears to induce IR through multiple mechanisms (Tremblay et al. 2005, Balage et al. 2011).

Currently, there is doubt that apoptosis is a major β-cell death mechanism in T2D. We found no indication that leucine affects INS-1E cell death at the concentrations of 1 and 5 mM (Fig. 5A), results that are in agreement with Zhang et al. (2009). This was further supported by the gene expressions of key mediators in apoptosis using RT-PCR. Thus, 5 mM leucine did not change Casp3 (Fig. 4A) and Dapk3 (Fig. 4B) expressions in INS-1E cells. Moreover, leucine significantly downregulated Bmf expression in INS-1E cells (Fig. 4C), suggesting an anti-apoptosis effect of leucine. The amount of β-cell mass, at any given moment, is represented by the sum of replication, size, and neogenesis minus the rate of apoptosis. The 3H-thymidine incorporation experiment showed that INS-1E cell proliferation was obviously reduced by exposure to elevated leucine levels for 72 h (Fig. 5B). This suggests that elevated leucine may cause reduced β-cell mass by decreasing β-cell mitotic division and proliferation.

In conclusion, chronic exposure to elevated levels of leucine induces β-cell dysfunction, with increased BIS and decreased GSIS in both isolated mouse islets and clonal β cells. Accordingly, long-term exposure of INS-1E cells to excess leucine resulted in abnormal CH metabolism, impaired OxPhos, and insulin signal transduction. Overall, these observations indicate that exposure to leucine may have detrimental effects on both β-cell function and insulin sensitivity.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work is carried out as a part of the research program of the Danish Obesity Research Centre (DanORC, see www.danorc.dk). The study was supported by The China Scholarship Council, The Daloon foundation, The TOYOTA-Foundation, Denmark, Institute of Experimental Clinical Research, Aarhus University, and the Nordic Centre of Excellence Programme (Systems biology in controlled dietary interventions and cohort studies-SYSDIET, P no. 070014).

Author contribution statement

Z L, P B J, S G, and K H designed this project, Z L completed experiments described in this manuscript. L B L completed proteomics experiment. P B J and S G provided experimental advice. Z L wrote the manuscript; K H, P B J, S G, and L B L helped with manuscript revision.

Acknowledgements

The authors thank Dorthe Rasmussen, Lene Trudsø, Kirsten Eriksen, Hanne Søndergaard Møller, and Tove Skrumsager Hansen for skilled technical assistance and Michael Væth (Department of Biostatistics, Aarhus University) for statistical advice.

References

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    (A) Effects of 72-h incubation with leucine on the response of mouse islets to glucose. The insulin secretion from mouse islets at low (3.3 mM) (ΔP<0.05, ΔΔP<0.01) and high (16.7 mM) glucose concentrations (*P<0.05 and **P<0.01) for 1 h compared with control (0.38 mM leucine). This part includes three independent experiments. For each experiment, we applied the mean value from 8 to 11 islets for each condition. (B) Effects of 72-h incubation with leucine on the response of INS-1E cells to glucose. The insulin secretion from per 10 000 INS-1E cells at low (3.3 mM) (ΔP<0.05, ΔΔP<0.01) and high (16.7 mM) glucose concentrations (**P<0.01, ***P<0.001) for 1 h compared with control (0.38 mM leucine). This part includes three independent experiments. For each experiment, we applied the mean value from six samples for each condition.

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    Changes in gene expressions in INS-1E cells induced by 5 mM leucine. The following genes were investigated: Pdx1 (A), MafA (B), Ins1 (C), Ins2 (D), Acaca (E), Fasn (F), Srebf2 (G), Hmgcr (H), Mvd (I), Sqle (J), Ldlr (K), and Cox6a2 (L). *P<0.05, **P<0.01, ***P<0.001 vs control, ΔΔΔP<0.001 vs control. RNA extraction includes three experiments applying different passage number cells. RT-PCR was performed in six repeats.

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    (A) Effect of leucine on TG content of INS-1E cells. Changes in TG content per 10 000 INS-1E cells after 72-h incubation with leucine (1–10 mM). ΔΔΔP<0.001 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from six samples for each condition. (B) Effect of leucine on CH content of INS-1E cells. Changes in CH content per 10 000 INS-1E cells after 72-h incubation with 1–10 mM leucine, *P<0.05, **P<0.01 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from six samples for each condition.

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    Impact of leucine on the expressions of genes associated with apoptosis. Alterations of mRNA expression in genes associated with apoptosis in INS-1E cells induced by 72 h exposure to 5 mM leucine. Casp3 (A), Dapk3 (B), Bmf (C), ΔΔΔP<0.001 vs control. RNA extraction includes three independent experiments. RT-PCR was performed in six repeats.

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    (A) Impact of leucine on the viability of INS-1E cells. Impact of 72-h incubation with leucine (1–10 mM) on the viability of INS-1E cells, ***P<0.001 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from ten samples for each condition. (B) Effect of leucine on the incorporation of 3H-thymidine into INS-1E cells. Effect of 72-h incubation with leucine (1–10 mM) on 3H-thymidine incorporation in INS-1E cells, ΔΔP<0.01, ΔΔΔP<0.001 vs control. This part includes three independent experiments. For each experiment, we applied the mean value from ten samples for each condition.

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    Scheme depicting major pathways relating to the possible effects of leucine on pancreatic β cells. AMPK, AMP-activated protein kinase; CH, cholesterol; CRMP-2, collapsin response mediator protein 2; ISGs, insulin secretory granules; OxPhos, oxidative phosphorylation; Srebf2, sterol-regulatory element binding factor 2; TCA, tricarboxylic acid. Dashed line arrow indicates negative effect, solid line arrow indicates positive effect.

  • AnelloMUcciardelloVPiroSPatanéGFrittittaLCalabreseVGiuffrida StellaAMVigneriRPurrelloFRabuazzoAM2001Chronic exposure to high leucine impairs glucose-induced insulin release by lowering the ATP-to-ADP ratio. American Journal of Physiology Endocrinology and Metabolism281E1082E1087.

    • Search Google Scholar
    • Export Citation
  • ArimuraNHattoriAKimuraTNakamutaSFunahashiYHirotsuneSFurutaKUranoTToyoshimaYYKaibuchiK2009CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity. Journal of Neurochemistry111380390. (doi:10.1111/j.1471-4159.2009.06317.x)

    • Search Google Scholar
    • Export Citation
  • BalageMDupontJMothe-SatneyITesseraudSMosoniLDardevetD2011Leucine supplementation in rats induced a delay in muscle IR/PI3K signaling pathway associated with overall impaired glucose tolerance. Journal of Nutritional Biochemistry22219226. (doi:10.1016/j.jnutbio.2010.02.001)

    • Search Google Scholar
    • Export Citation
  • CorlessMKielyAMcClenaghanNHFlattPRNewsholmeP2006Glutamine regulates expression of key transcription factor, signal transduction, metabolic gene, and protein expression in a clonal pancreatic β-cell line. Journal of Endocrinology190719727. (doi:10.1677/joe.1.06892)

    • Search Google Scholar
    • Export Citation
  • CunninghamGAMcClenaghanNHFlattPRNewsholmeP2005l-alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic β-cell line and protects from pro-inflammatory cytokine-induced apoptosis. Clinical Science109447455. (doi:10.1042/CS20050149)

    • Search Google Scholar
    • Export Citation
  • Del GuerraSLupiRMarselliLMasiniMBuglianiMSbranaSTorriSPolleraMBoggiUMoscaF2005Functional and molecular defects of pancreatic islets in human type 2 diabetes. Diabetes4727735. (doi:10.2337/diabetes.54.3.727)

    • Search Google Scholar
    • Export Citation
  • DikovskayaDLiZNewtonIPDavidsonIHutchinsJRKalabPClarkePRNäthkeIS2010Microtubule assembly by the Apc protein is regulated by importin-β–RanGTP. Journal of Cell Science123736746. (doi:10.1242/jcs.060806)

    • Search Google Scholar
    • Export Citation
  • DochertyHMHayCWFergusonLABarrowJDurwardEDochertyK2005Relative contribution of PDX-1, MafA and E47/β2 to the regulation of the human insulin promoter. Biochemical Journal389813820. (doi:10.1042/BJ20041891)

    • Search Google Scholar
    • Export Citation
  • El-AssaadWJolyEBarbeauASladekRButeauJMaestreIPepinEZhaoSIglesiasJRocheE2010Glucolipotoxicity alters lipid partitioning and causes mitochondrial dysfunction, cholesterol, and ceramide deposition and reactive oxygen species production in INS832/13 β-cells. Endocrinology15130613073. (doi:10.1210/en.2009-1238)

    • Search Google Scholar
    • Export Citation
  • FerranniniEGastaldelliAMiyazakiYMatsudaMPettitiMNataliAMariADeFronzoRA2003Predominant role of reduced β-cell sensitivity to glucose over insulin resistance in impaired glucose tolerance. Diabetologia4612111219. (doi:10.1007/s00125-003-1169-6)

    • Search Google Scholar
    • Export Citation
  • IshikawaMIwasakiYYatohSKatoTKumadakiSInoueNYamamotoTMatsuzakaTNakagawaYYahagiN2008Cholesterol accumulation and diabetes in pancreatic β-cell-specific SREBP-2 transgenic mice: a new model for lipotoxicity. Journal of Lipid Research4925242534. (doi:10.1194/jlr.M800238-JLR200)

    • Search Google Scholar
    • Export Citation
  • KalogeropoulouDLafaveLSchweimKGannonMCNuttallFQ2008Leucine, when ingested with glucose, synergistically stimulates insulin secretion and lowers blood glucose. Metabolism5717471752. (doi:10.1016/j.metabol.2008.09.001)

    • Search Google Scholar
    • Export Citation
  • KangDHGhoYSSuhMKKangCH2002Highly sensitive and fast protein detection with coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bulletin of the Korean Chemical Society2315111512. (doi:10.5012/bkcs.2002.23.11.1511)

    • Search Google Scholar
    • Export Citation
  • KruitJKWijesekaraNFoxJEMDaiXQBrunhamLRSearleGJMorganGPCostinAJTangRBhattacharjeeA2011Islet cholesterol accumulation due to loss of ABCA1 leads to impaired exocytosis of insulin granules. Diabetes6031863196. (doi:10.2337/db11-0081)

    • Search Google Scholar
    • Export Citation
  • KruitJKWijesekaraNWestwell-RoperCVanmierloTde HaanWBhattacharjeeATangRWellingtonCLLütJohannDJohnsonJD2012Loss of both ABCA1 and ABCG1 results in increased disturbances in islet sterol homeostasis, inflammation, and impaired β-cell function. Diabetes61659664. (doi:10.2337/db11-1341)

    • Search Google Scholar
    • Export Citation
  • LacyPEKostianovskyM1967Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes163539.

  • LarsenLBWedholm-PallasALindmark-MånssonHAndrénA2010Different proteomic profiles of sweet whey and rennet casein obtained after preparation from raw versus heat treated skimmed milk. Dairy Science & Technology90641656. (doi:10.1051/dst/2010024)

    • Search Google Scholar
    • Export Citation
  • LeeAKYeung-Yam-WahVTseFWTseA2011Cholesterol elevation impairs glucose-stimulated Ca(2+) signaling in mouse pancreatic β-cells. Endocrinology15233513361. (doi:10.1210/en.2011-0124)

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