Activation of Liver X Receptors and Retinoid X Receptors Induces ...

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Endocrinology 148(4):1843–1849 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1247

Activation of Liver X Receptors and Retinoid X Receptors Induces Growth Arrest and Apoptosis in Insulin-Secreting Cells Wolf Wente, Martin B. Brenner, Heike Zitzer, Jesper Gromada, and Alexander M. Efanov Lilly Research Laboratories, D-22419 Hamburg, Germany Liver X receptors (LXRs) form functional heterodimers with the retinoid X receptors (RXRs) and regulate cholesterol, lipid, and glucose metabolism. We demonstrated previously that activation of LXR modulates insulin secretion in MIN6 cells and pancreatic islets. In this study we investigated the effects of the LXR agonist T0901317 and the RXR agonist 9-cisretinoic acid (9cRA) on cell proliferation and apoptosis in MIN6 cells. Whereas T0901317 showed no effect on proliferation of MIN6 cells, combination of T0901317 with 9cRA inhibited cell proliferation. Flow cytometry analysis of cell cycle demonstrated that activation of LXR/RXR prevented MIN6 cells from G1 to G2 phase progression. Combination of T0901317 and 9cRA increased apoptosis rate and caspase-3/7

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IVER X RECEPTORS (LXRs) belong to the superfamily of nuclear hormone receptors. Members of the nuclear hormone receptor family are transcription factors, the activity of which is regulated by lipophilic small molecules such as steroid hormones, thyroid hormones, and vitamin D3 (1). Nuclear hormone receptors link physiological and nutritional signals with changes in gene expression. LXR forms heterodimeric complexes with their binding partner, retinoid X receptor (RXR) (2). LXR/RXR heterodimers regulate gene expression by preferential binding to the LXR response elements, double-stranded oligonucleotide direct repeats having the consensus sequence AGGTCA and separated by four nucleotides (3). Two receptors, LXR␣ and LXR␤, comprise the LXR family. LXR␣ is abundant in liver, adipose tissue, intestine, kidney, and spleen, whereas LXR␤ is expressed ubiquitously (4). Oxysterols, products of cholesterol hydroxylation, were found to be specific and physiological ligands for LXRs. LXRs respond to elevated cellular cholesterol by activation of expression of genes essential for removing cholesterol and other lipids from cells (4). In addition to regulation of cholesterol homeostasis, LXR modulates lipid and carbohydrate metabolism (5). Activation of LXR decreases blood glucose levels in a number of diabetic animal models (6). The effects of LXR activation on First Published Online December 28, 2006 Abbreviations: 7-AAD, 7-Amino-actinomycin D; BrdU, 5-bromo-2⬘deoxyuridine; 9cRA, 9-cis retinoic acid; FBS, fetal bovine serum; LXR, liver X receptor; RAR, retinoid acid receptor; RXR, retinoid X receptor; Smad3, mothers against decapentaplegic homolog 3. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

activity in MIN6 cells. Moreover, T0901317 or its combination with 9cRA significantly increased the cell susceptibility to free fatty acid- and cytokine-induced apoptosis. Treatment of MIN6 cells with LXR and RXR agonists produced a strong increase in expression of mothers against decapentaplegic homolog 3, a protein known to inhibit cell cycle G1/S phase progression and induce apoptosis. In isolated rat islets, the effect of palmitic acid on caspase-3/7 activity was increased with T0901317 alone and even more with the combination of T0901317 and 9cRA. Thus, activation of LXR/RXR signaling inhibits cell proliferation and induces apoptosis in pancreatic ␤-cells. (Endocrinology 148: 1843–1849, 2007)

blood glucose are mediated via both increases in insulin sensitivity and insulin secretion (6 – 8). We have shown previously that LXR␤ activation in pancreatic ␤-cells increases insulin secretion and insulin biosynthesis (9). The increase in insulin secretion is mediated via modulation of glucose/ lipid metabolism and is accompanied by changes in expression of genes crucial for the ␤-cell phenotype (9, 10). The number of pancreatic ␤-cells in the body is controlled by the balance between processes of neogenesis/proliferation and apoptosis. Cellular proliferation was suggested to be the major physiological mechanism for the ␤-cell replenishment as adult terminally differentiated ␤-cells maintain a significant proliferative capacity (11). Elevated ␤-cell apoptosis in type 2 diabetes is a major reason for the decrease in the ␤-cell number in the disease state (12). LXRs have been implicated in regulation of apoptosis in macrophages in bacterial infection settings (13, 14). LXR agonists were also shown to inhibit cellular proliferation in vascular smooth muscle cells, breast carcinoma, and prostate cancer cells (15– 17). We previously observed that whereas LXR agonist T0901317 increased insulin secretion, the combination of T0901317 with the RXR agonist 9-cis retinoic acid (9cRA) produced deterioration of pancreatic ␤-cell function (9). In this study we investigated effects of LXR/RXR activation on cellular proliferation and apoptosis in insulin-secreting cells. Materials and Methods Materials T0901317 was obtained from Cayman Chemicals (Ann Arbor, MI). 9cRA, 22(R)-hydroxycholesterol, propidium iodide, palmitic acid, and cytokines were from Sigma-Aldrich (St. Louis, MO), and caspase-3 inhibitor Z-DEVD-FMK was from Merck Biosciences (Nottingham, UK).

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Endocrinology, April 2007, 148(4):1843–1849

Culture of pancreatic islets and MIN6 cells Pancreatic islets were isolated from male Wistar rats (250 –300 g; Harlan, Horst, The Netherlands) as described (18). Islets were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen). Mouse insulinoma MIN6 cells (passages 25– 40) were cultured in DMEM medium (Invitrogen) containing 25 mm glucose, 50 ␮m 2-mercaptoethanol, 10% (vol/vol) heat-inactivated FBS (Sigma-Aldrich), 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen) as described (19).

Measurements of cellular proliferation MIN6 cells were seeded in a 96-well plate (7500 cells per well). The next day cells were starved for 24 h in glucose- and serum-free DMEM medium supplemented with 0.1% BSA. Then cells were incubated for 48 h in DMEM medium in the absence or presence of 10% FBS and test compounds, 10 ␮m 9cRA or 1 ␮m T0901317. During the last 5 h of this incubation, 5-bromo-2⬘-deoxyuridine (BrdU) solution was added to the incubation medium. The cellular proliferation was measured with cell proliferation ELISA BrdU assay (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer’s instructions.

Flow cytometry DNA content analysis MIN6 cells were incubated in DMEM medium with 1 ␮m T0901317 and 10 ␮m 9cRA for 48 h. Cells were harvested, washed with the PBS buffer (with 0.1% BSA), and fixed in ice-cold ethanol overnight. The next day cells were washed twice with PBS and incubated in a propidium iodide solution (PBS with 0.1% BSA and 50 ␮g/ ml propidium iodide) and 0.5 ␮g/ml DNase-free RNase (Roche Diagnostics) for 30 min. Propidium iodide staining was detected with the cytometer Guava PCA system (Guava Technologies, Hayward, CA). Data were plotted and analyzed using Guava CytoSoft software (Guava Technologies).

Annexin V binding Annexin binding to the cell membrane was assessed with Nexin reagent kit (Guava Technologies). Apoptosis was assessed after treating of MIN6 cells with test substances for 48 h in DMEM medium. Cells were harvested and resuspended in 1 ml of the Nexin buffer solution, 1 ␮l of phycoerythin conjugated annexin V (annexin V-PE) solution, and 5 ␮l 7-amino-actinomycin D (7-AAD) solution. Cells were cultured at 4 C for 1 h, washed, and analyzed with the cytometer Guava PCA system (Guava Technologies). Annexin V-PE fluorescence was detected by photomultiplier tube 1 (PM1), and 7-AAD fluorescence was detected by photomultiplier tube 2 (PM2).

Measurements of caspase activity and DNA fragmentation MIN6 cells were seeded in a 96-well plate (20,000 cells/well). The next day treatment of cells with test substances was started. Cells were incubated for 48 h in DMEM medium, and then DNA fragmentation and caspase activation assays were performed. Cytokine mixture used for apoptosis induction consisted of 20 ng/ml IL-1␤ and 40 ng/ml TNF-␣. Caspase-3/7 activity was measured with Apo-ONE caspase assay (Promega, Madison, WI) as an increase in the rhodamine 110 fluorescence (excitation 499 nm and emission 521 nm) after cleavage of the caspase substrate Z-DEVD-rhodamine 100. DNA fragmentation was measured as a accumulation of mono- and oligonucleosomes in the cytoplasmatic fraction of cells undergoing apoptosis with Cell Death ELISA Plus assay (Roche Diagnostics). To measure caspase-3/7 activation in isolated islets, groups of 100 islets were selected and incubated for 48 h with test compounds. After incubation islets were washed and transferred into a 96-well plate (10 islet/well) for caspase activity determination.

Total RNA extraction and real-time quantitative PCR Total RNA was extracted using the RNeasy microkit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. MIN6 cells or islets were disrupted in a buffer containing guanidine isothiocyanate and homogenized by vortexing for 1 min. RNA was then selectively bound by a silica-gel membrane, contaminants were washed

away, DNA was digested via column DNase treatment (RNase-Free DNase set; QIAGEN), and total RNA was eluted in RNase-free water. The concentration and quality of the RNA preparations were checked by spectrophotometry. Two hundred nanograms of RNA were reverse transcribed in a 50-␮l reaction mixture using the high-capacity cDNA archive kit (Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s instructions. Real-time PCR was performed using the ABI prism 7900 HS sequence detection system (Applied Biosystems). The PCR for each mRNA was performed in quadruple on cDNA samples in an ABI PRISM 384-well optical reaction plate (Applied Biosystems). A master mixture for each quadruple was set up in a total reaction volume of 30 ␮l: 5 ␮l cDNA (in a 1:5 dilution), 15 ␮l 2 ⫻ TaqMan Universal Master Mix, No AmpErase UNG (Applied Biosystems, Foster City, CA), 5 ␮l of diethylpyrocarbonate-treated water (Ambion Ltd., Huntingdon, UK), and 1.5 ␮l 20 ⫻ primer/probe set (Assay-on-Demand assay mix, Applied Biosystems). From this mixture an aliquot of 5 ␮l was placed in each of the four wells. The thermal cycling conditions were: 95 C for 10 min and then 40 cycles of 95 C for 15 sec and 60 C for 1 min. Relative expression was calculated by normalization to 36B4 mRNA by the ⌬⌬Ct method (20).

Immunoblotting MIN6 cells were lysed in the radioimmunoprecipitation buffer [50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% Na-deoxycholate, 5 mm EDTA, 0.1% sodium dodecyl sulfate] containing the Complete protease inhibitor cocktail (Roche Diagnostics). Protein samples with equal amounts of protein were separated by SDS-PAGE, transferred to polyvinylidine fluoride membrane, and probed with antibodies against mothers against decapentaplegic homolog 3 (Smad3; Santa Cruz Biotechnology, Santa Cruz, CA) and actin (Sigma-Aldrich). For signal quantification the VersaDoc imaging system (Bio-Rad, Munich, Germany) and the Quantity One 1-D Analysis Software 4.4 (Bio-Rad) were used.

Statistical analysis Results are presented as mean values ⫾ sem for indicated number of experiments. Statistical significances were evaluated using Mann-Whitney U test and Dunnett’s test for multiple comparisons using JMP 5 statistics software (SAS Institute, Cary, NC).

Results

To study cellular proliferation in insulin secreting MIN6 cells, we used BrdU incorporation assay. Treatment of cells for 48 h with either LXR agonist T0901317 (1 ␮m) or RXR agonist 9cRA (10 ␮m) did not significantly modify cellular proliferation in the presence of 10% FBS (Fig. 1A). However, when added to the culture medium together, T0901317 and 9cRA significantly inhibited BrdU incorporation by 50%. To better characterize the effect of LXR and RXR agonists on cellular proliferation, we analyzed effects of LXR/RXR activation on the cell cycle profile with flow cytometry after staining MIN6 cells with propidium iodide. Treatment of cells for 48 h with combination of T0901317 and 9cRa prevented cells progressing from G1 to G2 phase. The number of cells in G2 phase was 18.3 ⫾ 1.5 vs. 14.3 ⫾ 1.4% in vehicle and the T0901317/9cRA-treated group, respectively (P ⬍ 0.05, n ⫽ 6, Fig. 1B). In addition, LXR and RXR agonists induced a significant accumulation of cells in sub-G1 phase: 20.7 ⫾ 1.8 vs. 14.9 ⫾ 0.9% in vehicle and T0901317/9cRA-treated group, respectively (P ⬍ 0.05, n ⫽ 6, Fig. 1B). Accumulation of cells harboring subdiploid DNA is indicative of cells undergoing apoptosis. Apoptosis in MIN6 cells was examined with flow cytometry using annexin V and 7-ADD staining (21). Annexin V, an apoptosis marker, binds to phosphatidylserine, which

Wente et al. • LXR and Apoptosis in Pancreatic ␤-Cells

FIG. 1. T0901317 and 9cRA inhibit cellular proliferation in MIN6 cells. A, Effects of 1 ␮M T0901317, 10 ␮M 9cRA, and their combination on cellular proliferation measured as BrdU incorporation. Cells were treated for 48 h with vehicle (blank bar), 1 ␮M T0901317 (black bar), 10 ␮M 9cRA (hatched bar), and 1 ␮M T0901317 with 10 ␮M 9cRA (crossed bar). Data are mean ⫾ SEM for eight observations. ***, P ⬍ 0.001 vs. BrdU incorporation in the control group. B, DNA content histograms of MIN6 cells incubated in either the absence (vehicle) or combined presence of 1 ␮M T0901317 and 10 ␮M 9cRA for 48 h and then stained with propidium iodide. Data are representative of six observations.

translocates to the outer layer of the plasma membrane during apoptosis progression, whereas 7-AAD is a cell nonpermeable DNA intercalating dye used for dead cell discrimination. In the vehicle-treated group, only a small number of cells showed strong annexin V or 7-AAD staining (Fig. 2A). Treatment of cells with 1 ␮m T0901317 and 10 ␮m 9cRA resulted in the increased number of cells displaying strong annexin V and 7-AAD fluorescence (Fig. 2B). Cells exhibiting elevated annexin V staining are considered to be apoptotic, whereas cells with elevated annexin V and 7-AAD staining are undergoing secondary necrosis. Combination of T09013117 and 9cRA produced almost a 3-fold increase in apoptotic cells and doubling of cells undergoing secondary necrosis (Fig. 2C). Importantly, a very small number of cells with only increased 7-AAD fluorescence, reflecting cells undergoing primary necrosis, was detected at any tested condition (Fig. 2, A and B). Further characterization of apoptosis induction with LXR and RXR agonists was performed by measuring caspase-3/7 activity and DNA fragmentation. Treatment of MIN6 cells with 1 ␮m T0901317 for 48 h did not increase caspase-3/7 activity. However, in the presence of 9cRA, palmitic acid, or cytokines T0901317 significantly elevated caspase-3/7 activity (Fig. 3A). Potentiation of 9cRA-induced caspase-3/7 activity by T0901317 was concentration dependent with the LXR agonist displaying EC50 of 50 ⫾ 6 nm (data not shown).

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FIG. 2. Flow cytometry analysis of apoptosis. MIN6 cells have been treated with either vehicle (A) or a combination of 1 ␮M T09013117 and 10 ␮M 9cRA (B) for 48 h and then stained with annexin V and 7-AAD. Data are representative of four observations. C, Percentage of MIN6 cells undergoing apoptosis [Annexin(⫹) 7-AAD(⫺)]or secondary necrosis [Annexin(⫹) 7-AAD(⫹)] after treatment with either vehicle (blank bars) or the combination of 1 ␮M T0901317 and 10 ␮M 9cRA (black bars). Data are mean ⫾ SEM for four observations. *, P ⬍ 0.05 vs. corresponding control group.

Similar effects of T0901317 have been observed when examining the effects of the compound on DNA fragmentation, a hallmark of apoptosis. Whereas 1 ␮m T0901317 did not induce DNA fragmentation in cultured MIN6 cells, combination of T0901317 with either 9cRA or palmitic acid led to a significant increase in DNA breakdown (Fig. 3B). The caspase-3 inhibitor Z-DEVD-FMK (50 ␮m) blocked induction of caspase activity and DNA fragmentation produced with combination of palmitic acid and LXR/RXR agonists (Fig. 4, A and B). We also examined effects of other LXR agonists on caspase-3/7 activity. Naturally occurring cholesterol metabolite, 22(R)-hydroxycholesterol, is a potent LXR agonist (22). Treatment of MIN6 cells for 48 h with 10 ␮m 22(R)-hydroxycholesterol induced only a slight increase in caspase-3/7 activity (Fig. 5). When combined with 9cRA or palmitic acid, 22(R)-hydroxycholesterol induced a strong increase in caspase-3/7 activity similar to the effect of T0901317. Another synthetic LXR agonist GW3965 was also analog to T0901317 in caspase-3/7 induction in the presence of 9cRA and palmitic acid (data not shown). We explored the time course for DNA fragmentation induced with combination of LXR and RXR agonists. LXR/ RXR activation resulted in a slight increase in DNA breakdown after 24 h treatment (Fig. 6). The DNA breakdown induced by LXR and RXR agonists was further increased at 48 and 72 h treatment. In contrast, cytokine mixture of IL-1␤

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Wente et al. • LXR and Apoptosis in Pancreatic ␤-Cells

FIG. 5. Endogenous LXR agonist 22(R)-hydroxycholesterol induces caspase-3/7 activity. MIN6 cells have been treated for 48 h with either vehicle (blank bars) or 10 ␮M 22(R)-hydroxycholesterol (black bars) in the presence of 10 ␮M 9cRA, 1 mM palmitic acid (Palm), or their combination. Data are mean ⫾ SEM for six observations. **, P ⬍ 0.01 and ***, P ⬍ 0.001 vs. caspase activity in corresponding control group.

and TNF-␣ produced elevation in DNA fragmentation already at 4 h treatment, and the maximum effect of cytokines on DNA fragmentation was observed at 24 h treatment (Fig. 6). The slow development of the DNA breakdown observed with combination of T091317 and 9cRA suggests that induction of gene expression and protein synthesis is critical for apoptosis stimulation with LXR and RXR agonists.

Effects of LXR/RXR activation on mRNA expression of genes known to control G1/S transition were studied (Fig. 7A). Interestingly, treatment of MIN6 cells with T0901317 and 9cRA for 48 h produced a strong 8-fold increase in mRNA levels of Smad3, a protein known to inhibit cell cycle progression from G1 to S phase (23). In addition, small but significant increases of mRNA levels for Smad4 and cyclindependent kinase inhibitor p21Cip1 were observed in cells treated with LXR and RXR agonists (Fig. 7A). Effects on T0901317 and 9cRA on Smad3 mRNA and protein levels were explored in details. Treatment of MIN6 cells with either T0901317 or 9cRA produced significant 2-fold increases in Smad3 mRNA, whereas combination of both ligands produced synergistic elevation in the Smad3 mRNA levels (8fold; Fig. 7B). Either T0901317 or 9cRA treatment did not significantly change Smad3 protein levels (Figs. 7, C and D). Combination of both ligands elevated Smad3 protein levels in MIN6 by 43% (Fig. 7D).

FIG. 4. Caspase-3 inhibitor blocks caspase-3/7 activation and DNA fragmentation induced by LXR/RXR activation. MIN6 cells have been cultured for 48 h with vehicle (blank bars) or combination of 1 ␮M T09013117, 10 ␮M 9cRA, and 1 mM palmitatic acid in the absence (black bars) or presence of 50 ␮M Z-DEVD-FMK (hatched bars). Caspase-3/7 activity (A) and DNA fragmentation (B) were assessed at the end of the treatment. Data are mean ⫾ SEM for eight observations. ***, P ⬍ 0.001 vs. caspase activity or DNA fragmentation in corresponding control group.

FIG. 6. Time course of DNA fragmentation induced with cytokine cocktail or combination of LXR and RXR agonists. MIN6 cells have been treated for indicated time with vehicle (F), cytokines IL-1␤ and TNF-␣ (), or T0901317 and 9cRA (f). Data are mean ⫾ SEM for seven observations. *, P ⬍ 0.05; **, P ⬍ 0.01; and ***, P ⬍ 0.001 vs. DNA fragmentation at corresponding time in the control group.

FIG. 3. Effects of LXR and RXR agonists on caspase-3/7 activation and DNA fragmentation. MIN6 cells have been cultured for 48 h with either vehicle (blank bars) or 1 ␮M T09013117 (black bars) in the presence of 10 ␮M 9cRA, 1 mM palmitatic acid (Palm), or IL-1␤ with TNF-␣ (cytokines) as indicated. Caspase-3/7 activity (A) and DNA fragmentation (B) were assessed at the end of the treatment. Data are mean ⫾ SEM for six observations. *, P ⬍ 0.05 and ***, P ⬍ 0.001 vs. caspase activity or DNA fragmentation in corresponding control group.

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FIG. 8. Effects of LXR and RXR agonists on caspase-3/7 activation and Smad3 mRNA in isolated rat pancreatic islets. A, Caspase-3/7 activity in rat islets treated for 48 h with vehicle (blank bars) or 1 mM palmitic acid (black bars) in the presence of 1 ␮M T0901317 (T0) and 10 ␮M 9cRA as indicated. B, Relative Smad3 mRNA expression in rat islets treated with vehicle (blank bar) or combination of 1 ␮M T0901317 and 10 ␮M 9cRA for either 24 h (black bar) or 48 h (hatched bar). Data are mean ⫾ SEM for eight (A) and four (B) observations. *, P ⬍ 0.05 vs. caspase activity or mRNA levels in corresponding control group.

FIG. 7. LXR/RXR activation increases Smad3 expression. A, Relative mRNA expression of genes controlling G1/S phase progression in MIN6 cells treated with either vehicle (blank bars) or 1 ␮M T0901317 with 10 ␮M 9cRA (black bars). Data are mean ⫾ SEM for four observations. *, P ⬍ 0.05 vs. expression in vehicle-treated cells. B, Relative Smad3 mRNA expression in MIN6 cells treated with vehicle (blank bar), 1 ␮M T0901317 (hatched bar), 10 ␮M 9cRA (crossed bar), or their combination (black bar). Data are mean ⫾ SEM for four observations. *, P ⬍ 0.05 vs. expression in vehicle-treated cells. C, Smad3 protein levels in MIN6 cells treated with vehicle (lane 1), 1 ␮M T0901317 (lane 2), 10 ␮M 9cRA (lane 3), or their combination (lane 4). Blot is representative of six different experiments. D, Relative Smad3 protein expression in MIN6 cells treated with vehicle (blank bar), 1 ␮M T0901317 (hatched bar), 10 ␮M 9cRA (crossed bar), or their combination (black bar). Data are mean ⫾ SEM for six observations. *, P ⬍ 0.05 vs. protein expression in vehicle-treated cells.

Effects of T0901317 on caspase activity were also assessed in primary nondividing pancreatic cells. Caspase-3/7 activation was measured in isolated rat pancreatic islets treated for 48 h with 1 ␮m T0901317 or combination of 1 ␮m T0901317 with 10 ␮m 9cRA in the presence of 1 mm palmitic acid (Fig. 8A). T0901317 did not increase caspase-3/7 activity under normal culture conditions. However, the LXR agonist further increased palmitic acid-induced elevation of caspase-3/7 activity. Combination of LXR and RXR agonists also aggravated effects of palmitic acid on caspase activity (Fig. 8A). Similar to MIN6 cells, treatment of rat islets with combination of LXR and RXR agonists for either 24 or 48 h produced a 2-fold increase in Smad3 mRNA expression (Fig. 8B). Discussion

We examined the effects of the activation of LXR/RXR heterodimers on cell growth and death in insulin-secreting

MIN6 cells and rat pancreatic islets. We determined that combination of the LXR agonist T0901317 and the RXR agonist 9cRA produced growth arrest and apoptosis induction in MIN6 cells. In rat islets T0901317 renders cells more sensitive to free fatty acid-induced apoptosis. Recent data suggest that LXR activation inhibits cellular proliferation in a number of cell lines (15–17). The inhibition of proliferation with LXR agonists in vascular smooth muscle cells was mediated via increased protein expression of the cyclin-dependent kinase inhibitor p27Kip1 and reduced expression of the S-phase kinase-associated protein 2 (15). Our results are in agreement with previously reported data showing antiproliferative effects of LXR agonists. Oxysterols were reported to cause apoptosis and induce growth arrest in a wide variety of cells (24). The precise mechanism for the oxysterol induction of apoptosis is unknown but has been suggested to involve activation of both extracellular and mitochondrial apoptotic pathways, stimulation of calcium influx, and activation of classical caspase cascade (25). No correlation between apoptosis induction and LXR potency for different oxysterols has been observed, and some oxysterols, potent LXR agonists, display only mild efficacy in apoptosis induction (26). In the current study, 22(R)-hydroxysterol behaved differently from T0901317 and already by itself induced caspase-3/7 activation, which confirms LXR-independent mechanisms for the oxysterol toxicity. However, 22(R)-hydroxysterol mimicked T0901317 in its synergy with 9cRA and palmitic acid on caspase-3/7 activation. Based on these data, it is tempting to speculate that LXR activation contributes to apoptosis induction for certain oxysterols. Retinoids play an important role in the control of differentiation and growth of a broad array of normal and tumor cell types by inhibition of cell proliferation or induction of differentiation and apoptosis (27). Both RXR and retinoid acid receptor (RAR) subtypes could mediate these effects. In our study we used a natural retinoid 9cRA known to activate both RXR and RAR subtypes (28). However, the synergistic effects of 9cRA observed in combination with T0901317 in

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MIN6 cells were mediated exclusively via RXR. The RAR selective agonist Am580 did not synergize with the LXR agonist in inducing apoptosis, whereas the RXR selective agonist methoprene replicated effects of 9cRA (data not shown). Growth arrest and apoptosis induction with LXR and RXR agonists as seen in the current study can be explained by stimulation of Smad3 expression. MIN6 cells treated with T0901317 and 9cRA induced a strong increase in the expression of Smad3 measured on both mRNA and protein levels. Smad3 is a member of the Smad protein family, pivotal intracellular mediators of the TGF-␤ receptor signaling (29). Upon binding of TGF-␤ to its receptor, Smad3 undergoes phosphorylation. Phosphorylated Smad3 forms complex with a copartner Smad4, and Smad heterodimers translocate into the nucleus, in which they regulate expression of the target genes in a cell type-specific fashion via interactions with transcriptional coactivators and corepressors (29). Smad3 inhibits cell cycle progression from G1 to S phase via regulation of expression of cyclin-dependent kinase inhibitors (23). Smad3 overexpression also promotes TGF-␤-induced apoptosis via regulation of expression and processing of the Bcl-2 family of proteins (30 –32). Although the mechanisms of Smad3 induction with LXR agonist are not clear, interaction between TGF-␤ signaling pathway and LXR␤ has been reported before (33). Interestingly, we observed synergy between palmitic acid and LXR/RXR activation in apoptosis induction, whereas cytokines and LXR/RXR agonists do not display such synergy. The reason for these effects is not clear but may imply that cytokines and LXR/RXR activate similar apoptosis signaling pathways. However, we did not see an increase in the nitric oxide synthase protein expression in MIN6 cells cultured with T0901317 and 9cRA as in contrast to the strong nitric oxide synthase induction upon cytokine treatment (data not shown). It is currently unknown whether LXR activity is elevated under conditions of the metabolic syndrome and type 2 diabetes. We did not observe any effect of high glucose concentrations on LXR protein levels in MIN6 cells (9). However, recent data indicate that high glucose may induce nuclear localization and activation of LXRs in pancreatic ␤-cells (34). In addition, islets from diabetic db/db mice display increased levels of LXR␤ and Smad3 mRNA (Zitzer, H., unpublished data). It remains to be seen whether at certain pathophysiological conditions, hyperactivation of the LXR/RXR pathway may contribute to the development of the pancreatic ␤-cell dysfunction. Acknowledgments Received September 11, 2006. Accepted December 20, 2006. Address all correspondence and requests for reprints to: Dr. Alexander M. Efanov, Lilly Research Laboratories, Essener Bogen 7, D-22419 Hamburg, Germany. E-mail: [email protected]. Present address for J.G.: Novartis Institute for Biomedical Research, Cambridge, Massachusetts. Disclosure Statement: W.W. has nothing to declare; H.Z., M.B.B., and A.M.E. hold stock in Eli Lilly; J.G. holds stock in Novartis, Eli Lilly, and Novo Nordisk.

Wente et al. • LXR and Apoptosis in Pancreatic ␤-Cells

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