Impact of PPAR{gamma} overexpression and activation on pancreatic islet gene expression profile analyzed with oligonucleotide microarrays

Laura E. Parton,1 Frédérique Diraison,1 Suzanne E. Neill,2 Sujoy K. Ghosh,2 Mark A. Rubino,2 John E. Bisi,2 Celia P. Briscoe,2 and Guy A. Rutter1

1Henry Wellcome Signalling Laboratories and Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD United Kingdom; and 2GlaxoSmithKline, Department of Metabolic Diseases, Research Triangle Park, North Carolina 27709

Submitted 9 January 2004 ; accepted in final form 26 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) serves as a target for the thiazolidinedione class of antidiabetic drugs and is an important regulator of adipose tissue differentiation. By contrast, the principal target genes for PPAR{gamma} in the pancreatic islet and the impact of their induction on insulin secretion are largely undefined. Here, we show that mRNAs encoding both isoforms of rodent PPAR{gamma}, {gamma}1 and {gamma}2, are expressed in primary rat islets and are upregulated by overexpresssion of the lipogenic transcription factor sterol response element-binding protein 1c. Unexpectedly, however, oligonucleotide microarray analysis demonstrates that graded activation of PPAR{gamma} achieved with 1) the thiazolidinedione GW-347845, 2) transduction with adenoviral PPAR{gamma}1, or 3) a combination of both treatments progressively enhances the expression of genes involved in fatty acid oxidation and transport. Moreover, maximal activation of PPAR{gamma}1 reduces islet triglyceride levels and enhances the oxidation of exogenous palmitate while decreasing glucose oxidation, cellular ATP content, and glucose-, but not depolarization-stimulated, insulin secretion. We conclude that, in the context of the pancreatic islet, the principal response to PPAR{gamma} expression and activation is the activation of genes involved in the disposal, rather than the synthesis, of fatty acids. Although fatty acid oxidation may have beneficial effects on {beta}-cell function in the longer term by countering {beta}-cell "lipotoxicity," the acute response to this metabolic shift is a marked inhibition of insulin secretion.

{beta}-cell; insulin; secretion; peroxisome proliferator-activated receptor-{gamma}; sterol regulatory element-binding protein-1c; thiazolidinedione; glucolipotoxicity


GROWING EVIDENCE SUGGESTS that {beta}-cell secretory failure in some forms of type 2 diabetes is correlated with the accumulation of triglyceride (TG) within the pancreatic islet (28). In the Zucker diabetic fatty (ZDF) rat, overabundance of islet lipid is associated with impaired glucose-stimulated insulin secretion and decreased expression of the glucose transporter GLUT2/Slc2a2 (25), as well as enhanced nitric oxide formation (42) and apoptosis (44). Although the mechanisms underlying these changes, collectively termed "lipotoxicity" or "glucolipotoxicity" (36) are not fully elucidated, elevated levels of "lipogenic" transcription factors, such as sterol response element-binding protein-1c (SREBP-1c) (26) and peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) (26, 28), may play a role.

PPAR{gamma}, a member of the nuclear hormone receptor family, is translated from an alternately spliced mRNA in humans and rodents, generating three ({gamma}1, {gamma}2, and {gamma}3) or two ({gamma}1 and {gamma}2) isoforms, respectively, in these species (see Fig. 1A) (16, 17, 45, 48). Whereas PPAR{gamma}1 is present in many mammalian tissues, the expression of PPAR{gamma}2 is confined largely to adipose tissue (48). Both PPAR{gamma} mRNA and protein have been detected in human (13) and rodent (4, 53) pancreatic islets, although the relative levels of the {gamma}1 and {gamma}2 isoforms have yet to be established in this tissue.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Effects of constitutively active sterol regulatory element-binding protein (SREBP CA) and glucose on peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) isoform gene expression as determined by real-time RT-PCR. A: organization of the mammalian PPAR{gamma} gene. Eight exons code for PPAR{gamma}1 (A1, A2, and 1–6); 7 exons code for PPAR{gamma}2 (B1 and 1–6). B and C: primary rat islets were treated for 32 h with SREBP CA or null adenovirus and then cultured in low glucose for 16 h overnight followed by a further 8 h in either 3 or 17 mM glucose. mRNA levels of PPAR{gamma}1 and -{gamma}2 were analyzed by semiquantitative RT-PCR (insets; 30 cycles) generating the expected 550-bp products as analyzed by agarose (1% wt/vol) gel electrophoresis and staining with ethidium bromide or quantitative real-time RT-PCR (histograms). Results are expressed as fold changes over control (null, 3 mM glucose) and presented as means + SE. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001. D: total RNA was extracted from MIN6 cells, and semiquantitative RT-PCR was performed using primers recognizing both PPAR{gamma} isoforms before gel electrophoresis.

 
PPAR{gamma} is a well-defined target of the thiazolidenedione (TZD) class of antidiabetic drugs (20, 41) and is likely to mediate the transcriptional effects of these agents in adipose tissue and muscle. On the other hand, loss of PPAR{gamma} function also exerts antidiabetic effects in some circumstances. Thus deletion of one allele of the mouse PPAR{gamma} gene prevents adipocyte hypertrophy and the development of insulin resistance in response to a high-fat diet (27). Moreover, an inactivating mutation of PPAR{gamma}2 (Pro12Ala) that occurs as a polymorphism in the human genome increases insulin sensitivity and decreases the risk of type 2 diabetes (21, 33).

Recent data have suggested that changes in PPAR{gamma} activity in the islet may also influence the development of diabetes. First, of four morbidly obese individuals carrying a rare activating mutation (Pro113Glu) of PPAR{gamma}2 (38), three were diabetic with decreased circulating insulin levels. Second, activation of PPAR{gamma}1 has been shown to inhibit glucose-stimulated insulin release ex vivo both from a tumoral {beta}-cell line (34) and from primary islets (3). Third, PPAR{gamma} expression is increased more than fivefold in islets from ZDF rats (53), suggesting that PPAR{gamma} induction may also negatively regulate insulin release in this model. In contrast to these observations, PPAR{gamma} activation has been suggested to preserve {beta}-cell function, morphology, and mass in other rodent models of diabetes. Thus TZDs, presumably acting by binding to PPAR{gamma} (29), reduce islet TG content and promote glucose- and arginine-stimulated insulin secretion in the fat-laden islets of ZDF rats (26, 43). Furthermore, TZD treatment enhances insulin release in glucose-intolerant human subjects (7). One possible explanation for these discrepancies is that changes in {beta}-cell function in vivo may not result from a direct effect of TZDs on the islet but may instead be a consequence of improved whole body metabolic parameters (e.g., blood glucose and circulating free fatty acid levels).

In light of such contrasting observations, the present study was designed to evaluate the effects of PPAR{gamma} activation and/or overexpression on 1) the gene expression profile, 2) glucose and fatty acid metabolism, and 3) glucose-stimulated insulin secretion in primary isolated rat islets. Using a combination of agonist treatment and adenoviral gene delivery to achieve a graded activation of PPAR{gamma}, we show that this factor unexpectedly enhances the expression of genes responsible for fatty acid oxidation and disposal in islets, including those involved in mitochondrial and peroxisomal {beta}-oxidation, fatty acid transport, and binding. Although the consequent metabolic changes are associated with an acute suppression of glucose oxidation and glucose-stimulated insulin release, they may also serve to preserve islet {beta}-cell mass in the longer term by attenuating the proapoptotic effects of fatty acids and their nonoxidative metabolites (43).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

Collagenase (1.2 pzu/mg) was obtained from Boehringer Mannheim (Mannheim, Germany). Culture medium (Dulbecco's modified Eagle’s medium; DMEM), and heat-inactivated fetal bovine serum (FCS), were obtained from Life Science Technologies (Paisley, UK). Histopaque gradient solutions, penicillin, streptomycin, glutamine, and TRI Reagent were obtained from Sigma (Poole, Dorset, UK).

Amplification of Recombinant Adenoviruses

Recombinant adenoviruses encoding 1) constitutively active SREBP-1c (amino acids 1–403, wild-type sequence; SREBP CA) (2, 18), 2) enhanced green fluorescent protein (eGFP; null), or 3) PPAR{gamma}1 (wild-type sequence; Ad.PPAR{gamma}), were generated and amplified as described (22).

Islet Isolation and Culture

Pancreatic islets were isolated from male Wistar rats (225–250 g) and killed according to locally approved animal procedures. Perfusion of the pancreatic duct and in situ collagenase digestion were performed as described (19). Islets were subsequently purified on either Histopaque gradient solutions (10 ml of 1.119 g/l; 6 ml of 1.083 g/l, 6 ml of 1.077 g/l) or gradients of bovine serum albumin (35%, 31% and 27% wt/vol). Isolated islets were cultured in suspension for 16 h in DMEM containing 10% (vol/vol) FCS, 11 mM glucose, 2 mM glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin and incubated at 37°C with 95% air-5% CO2. Islets were then hand picked the next day for further experiments.

Adenoviral Infection

Islets were infected with SREBP CA, Ad.PPAR{gamma}, or null adenoviruses at a multiplicity of infection of 50 viral particles/cell for 32 h. Islets were then placed in medium containing 3 mM glucose in the presence of the PPAR{gamma} agonist GW-347845 (200 nM) or its vehicle (dimethyl sulfoxide at a final concentration of 0.004%) for a further 16 h before use.

Western Blot Analysis

Total protein extracts (25 µg) were resolved by SDS-PAGE (10% bisacrylamide-acrylamide, vol/vol) and transferred to polyvinylidene difluoride membranes (50), followed by immunoblotting with rabbit polyclonal anti-PPAR{gamma} antibody raised against a peptide corresponding to amino acids 6–105 of PPAR{gamma} (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies were revealed using BM Chemiluminescence blotting substrate (Roche Diagnostics, Mannheim, Germany).

Immunocytochemistry

Islet slices were prepared essentially as described (12). Briefly, islets were fixed with Zamboni's fixative overnight at 4°C, and sections of islets (10 µm) were obtained using a cryostat (Bright OTF5000; Jencons, Leighton Buzzard, UK). Islets were permeabilized with 0.3% (vol/vol) Triton X-100 overnight and then incubated with primary antibodies at 4°C. Rabbit polyclonal anti-PPAR{gamma} antibody was used at 1:50 dilution and guinea pig anti-insulin antibody at 1:500 dilution. Primary antibodies were revealed using TRITC- or FITC-conjugated secondary antibodies against rabbit or guinea pig IgG (1:500 dilution), respectively.

Microarray Analysis

Sample preparation and processing. Total RNA samples from four separate islet cultures per condition were isolated by TRI Reagent and purified on an RNeasy column (Qiagen, Valencia, CA) before labeling for hybridization to Affymetrix RAE230 rat arrays (Affymetrix, Santa Clara, CA). Total RNA was prepared for hybridization and subsequently hybridized according to recommendations from Affymetrix (Genechip Expression Analysis Technical Manual [online] http://www.affymetrix.com/support/technical/manual/expression_manual.affx) (2003), based on a small-sample labeling protocol. Briefly, each sample was processed from 100 ng of total RNA that were first converted to cDNA. All of the cDNA was subsequently converted to cRNA in the first round of linear amplification. The resulting cRNA (400 ng) was used as a template for second-round cDNA synthesis that was again converted to cRNA. The final cRNA product (20 µg) was fragmented, and 15 µg of the fragmented cRNA were hybridized to the Affymetrix chips. Hybridized chips were washed and then scanned on an Affymetrix GeneChip 3000 confocal scanner. Gene expression data were generated in the GeneChip MAS 5.0 software from Affymetrix. Both total RNA and cRNA samples were checked for quality on an RNA 6000 Nano LabChip with the Bioanalyzer system (Agilent Technologies, Palo Alto, CA).

Microarray data analysis. From the GeneChip MAS 5.0 software, we obtained an expression signal and a Present/Absent call status for every probe set on each of the hybridized Affymetrix chips. Multiple probe sets often encode a single gene. All expression data were normalized by global scaling to a trimmed average intensity of 150 per chip. The Present/Absent call status was used as a criterion for gene filtering; probe sets with a Present call in three of the four replicates in at least one of the four treatments were kept for subsequent analysis. Control, noneukaryotic genes were also removed. After filtering, 9,563 of a total of 15,923 probe sets were retained for analysis.

A principal-components analysis was performed on the 16 (4 treatments x 4 replicates per treatment) samples, using gene signals as sample descriptors to identify outlier samples. None of the samples behaved as outliers (data not shown); consequently, the full set of 16 samples was retained for analysis.

Gene signals were logged (to base 2). The mean signal for all genes within replicate samples was calculated on the logged signals. Log ratios of the average signals were calculated to estimate the fold change in gene expression between treatments. Statistical significance of the difference in expression for a gene between two treatments was assessed by the heteroscedastic two-tailed Student's t-test for unpaired samples. Because the t-test was performed over thousands of genes, a false discovery rate (46) was also calculated to adjust for multiple testing using the Q-value program in the R statistical package.

Analysis of clusters. Genes were ranked in order of increasing P value according to differences between the PPAR{gamma} plus GW-347845 vs. null adenovirus groups. Among those genes that showed highly significant (P < 0.01) changes (~300 in total), clusters were identified for all groups (null, agonist alone, PPAR{gamma} alone, or combined treatment) according to expression changes (Gene Tree, GeneSpring; SiliconGenetics, Redwood City, CA; www.silicongenetics.com) (15) (see Fig. 3D) and functional similarity (GeneSpring and GeneMAPP, Gladstone Institutes, University of California at San Francisco, www.genemapp.org; see Table 2).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 3. Effects of PPAR{gamma} activation and/or overexpression on islet gene expression profiles. Impact of 200 nM GW-347845 (A), PPAR{gamma}1 overexpression (B), and GW-347845 + PPAR{gamma} expression (C) on gene expression profiles in cultured islets is illustrated. All mRNAs positively identified as present in ≥3 of 4 of the replicate microarrays from ≥1 condition are shown. D: gene tree cluster analysis (see MATERIALS AND METHODS) of the 300 mRNAs showing the most significant changes in response to PPAR{gamma}-infection + GW-347845. Levels of all genes in this group differed highly significantly (P < 0.01) from those in null virus-infected islets. Expression levels are represented in pseudocolor for islets infected with null virus only (1), treated with 200 nM GW-347845 only (condition A; 2), or infected with PPAR{gamma} virus and incubated in the absence (3) or presence (4) of agonist (conditions B and C, respectively). Three clustered regions (a–c) were apparent: clusters a and b (i) comprised mRNAs whose levels fell with treatment, b (ii) and c (ii) those that increased. The majority of genes in groups c (i) and c (ii) responded to all 3 conditions. Genes are identified by accession no.; spaces indicate no changes with respect to null virus condition.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Transcripts differentially regulated by PPAR{gamma}/GW-347845 in primary islets

 
The microarray data presented in this paper have been submitted to the National Center for the Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) website, GEO accession: gpl921.

Semiquantitative and Real-Time Quantitative RT-PCR

Total RNA was extracted using TRI Reagent from ~100 islets. RNA samples were treated with DNA-free (Ambion, Austin, TX) to remove any contaminating genomic DNA, and the concentration was determined using RiboGreen assay (Molecular Probes, Eugene, OR). Complementary DNA was synthesized from 1 µg of total RNA in 100 µl by using random hexamers and Moloney murine leukemia virus reverse transcriptase. All genes, primers, and probes used in real-time RT-PCR analysis are described in Table 1. PCR was performed using 20 ng of reverse-transcribed total RNA with 900 nM of sense and antisense primers, 5.5 mM MgCl2, 300 µM dNTP, 1.25 U of Taq polymerase and 1x Taqman buffer A (Applied Biosystems, Foster City, CA) in a total volume of 25 µl in an ABI PRISM 7700 Sequence Detection System instrument. Standard curves were constructed by amplifying serial dilutions of untreated rat islet cDNA (50 ng to 0.64 pg) and plotting cycle threshold values as a function of starting reverse-transcribed RNA, the slope of which was used to calculate relative expression of the target gene.


View this table:
[in this window]
[in a new window]
 
Table 1. Primer and probe sequences used in quantitative RT-PCR analysis

 
Intracellular TG Measurements

Total lipids were extracted from 150 islets by use of a chloroform-methanol (2:1 vol/vol) mixture. The extracted lipids were air dried, and 10 µl of a detergent (Thesit; Fluka, UK) were added to the dry pellet. Samples were air dried again and resuspended in 50 µl of water. TG was measured using a commercial kit (Infinity Triglyceride Reagent, Sigma) and a standard curve of triolein (Sigma) treated in parallel with the samples.

Insulin Secretion

Forty-eight hours after adenoviral infection, islets were incubated for 60 min at 37°C in 2 ml of Krebs-Ringer bicarbonate-HEPES buffer (KRBH) [in mM: 130 NaCl, 3.6 KCl, 1.5 CaCl2, 0.5 MgSO4, 0.5 KH2PO4, 2.0 NaHCO3, and 10 HEPES] supplemented with 3 mM glucose, 0.1% (wt/vol) bovine serum albumin preequilibrated with 95% O2-5% CO2, pH 7.4. Islets were separated into three groups of five islets per condition and incubated as above for 30 min in 1 ml of KRBH containing either 3 or 17 mM glucose or 3 mM glucose plus KCl (35 mM). Total insulin was extracted in acidified ethanol (1). Insulin was measured using radioimmunoassay by competition with 125I-labeled rat insulin (Linco Research, St. Charles, MO) according to the manufacturer's instructions. Insulin secretion was calculated as the percentage of total islet insulin content and normalized as the fold change between individual experiments. Typically, insulin release in the presence of 17 mM glucose represented 2–3% of total islet insulin content/30 min.

ATP Assay

Rat islets were prepared and incubated as described above for the assay of insulin secretion. After experimental manipulation, islets were lysed in ice-cold perchloric acid (20% vol/vol, 125 µl), rapidly frozen, and stored at –80°C. Samples were thawed and adjusted to pH 7.4 with a known volume of neutralization mixture (0.5 M triethanolamine, 2 M KOH, 100 mM EDTA). Precipitated potassium perchlorate was removed by centrifugation (10,000 g, 2 min) and the supernatant stored on ice before assay. Neutralized sample (10 µl) was added to 970 µl of assay buffer (130 mM NaHAsO4, 17 mM MgSO4, 4 µM NaH2PO4, pH 7.4) and 10 µg of firefly lantern extract (10 mg/ml stock, Sigma). The reaction was initiated with 10 µg of firefly luciferase, and light emission was recorded for 30 s using a photon counting luminometer. ATP concentration was obtained by comparison to standards (0–25 pmol) prepared in parallel with the cell extracts.

[1-14C]Palmitate Oxidation

Islets were preincubated for 30 min at 37°C in KRBH supplemented with 3 mM glucose and 0.5% fatty acid-free BSA (wt/vol). Triplicate groups of 100 islets were then placed in 250 µl of oxidation mix (KRHB containing 0.2 mM unlabeled palmitate, 1 mM carnitine, 0.4 µCi/ml [1-14C]palmitate, 0.5% fatty acid-free BSA and 3 or 17 mM glucose) in a 24-well plate. A rubber gasket the size of the 24-well plate and containing 0.5-cm holes was aligned over the plate. A UniFilter-24 GF/B Plate (PerkinElmer, Beaconsfield, UK) was sealed with an adhesive sheet, and 100 µl of 40% (wt/vol) KOH were pipetted onto each filter. The filter plate was inverted and aligned over the rubber gasket to form a small CO2 capture chamber. Finally, the chamber was sealed with a 6-mm glass plate, a 6-mm metal plate, and a lead weight to ensure an airtight seal. The apparatus was incubated for 2 h at 37°C. Filters were removed, and captured 14CO2 was measured by scintillation counting. Control incubations lacking islets were included in each incubation series.

[U-14C]Glucose Oxidation

Islets were preincubated for 30 min at 37°C in KRBH supplemented with 3 mM glucose and 0.5% BSA (wt/vol). Triplicate groups of 100 islets were then placed in 250 µl of KRHB containing 3 or 17 mM unlabeled glucose, 1.7 µCi [U-14C]glucose, and 0.5% BSA (wt/vol) in a 24-well plate. A CO2 capture chamber was created as above for palmitate oxidation and incubated for 2 h at 37°C. Captured 14CO2 was measured by scintillation counting.

Statistical Analysis

All functional analyses were performed at least three times in triplicate. Data are presented as means ± SE. Statistical significance was assessed by Student's t-test for unpaired comparison and two-tailed analysis.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PPAR{gamma}1 and -{gamma}2 Are Expressed in Pancreatic Islets and Are Downstream Targets of SREBP-1c

Consistent with earlier reports (13), mRNAs encoding both PPAR{gamma}1 and -{gamma}2 (Fig. 1A) were detectable in rat islets by semiquantitative and quantitative RT-PCR (Fig. 1, B and C). The expression of both isoforms was further induced ({gamma}1 by ~7-fold and {gamma}2 by ~2.5-fold; Fig. 1, B and C) by adenoviral transduction of islets with the active nuclear fragment of SREBP-1c (2, 11, 51). Confirming the likely expression of PPAR{gamma} in {beta}-cells within the islet: 1) primers able to amplify either isoform generated the expected product by PCR using clonal MIN6 {beta}-cell-derived cDNA (Fig. 1D), and 2) PPAR{gamma} immunoreactivity, significantly above that in slices incubated in the absence of primary antibody (not shown), was associated with insulin-containing structures in null adenovirus-infected islets (see below; Fig. 2B).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2. Adenovirus-mediated overexpression of PPAR{gamma} in pancreatic islets. Isolated islets were infected with null (Ad.Null)- or PPAR{gamma} (Ad.PPAR{gamma})-encoding adenoviruses and cultured for 32 h at 11 mM glucose before 16-h culture at 3 mM glucose and then 8-h culture in 3 or 17 mM glucose. Total protein extraction (A) or preparation of cryostat sections (B) was then performed. A: protein extracts (25 µg) were subjected to immunoblot analysis, and PPAR{gamma} was visualized with a PPAR{gamma} polyclonal antibody (see MATERIALS AND METHODS). B: cryostat sections were fixed and permeabilized at 4°C (16 h) in the presence of 0.3% (vol/vol) Triton X-100 in PBS. Sections were probed with anti-PPAR{gamma} rabbit polyclonal antibody or with guinea pig anti-insulin and revealed with TRITC with appropriate secondary antibodies. Immunolabeled islets were imaged on a Leica SP2 laser scanning confocal microscope using a x63 oil immersion objective with excitation at 488 nm (Ar) and 543 nm (He-Ne) laser for excitation. Emission was detected at >515 (green, PPAR{gamma}) and >560 nm (red, insulin). Scale bar, 50 µm.

 
Effects of PPAR{gamma}1 Overexpression on Gene Expression in Isolated Islets

In an effort to identify the principal target genes for PPAR{gamma} in the islet, we next explored the impact of adenoviral transduction of islets with PPAR{gamma}1. As shown in Fig. 2A, infection of islets with PPAR{gamma}1-bearing virus increased levels of PPAR{gamma} protein ~10-fold. PPAR{gamma}1 protein expression was detected in 30–75% of islet cells as assessed by immunocytochemical analysis of islet slices. Although low levels of PPAR{gamma} overexpression could be detected throughout both small (≤50 µm diameter) and larger islets (Fig. 2B, bottom), the highest levels were achieved in cells at the islet periphery, of which the majority were insulin positive (Fig. 2B, bottom, "zoom"). This pattern is similar to that previously reported in islets infected with either null (eGFP-expressing) or SREBP-1c-encoding adenoviruses (12) and was not associated with any detectable increase in cellular apoptosis (assessed by annexin V binding) or necrosis (12) at the viral titer deployed (data not shown).

Oligonucleotide microarray analysis (Fig. 3 and Table 2) revealed that, of ~15,000 mRNAs and expressed sequence tags present on the arrays used, 9,563 were present in islets (see MATERIALS AND METHODS). To test the effect on gene expression of the progressive activation of PPAR{gamma}, we employed three protocols: A) treatment with a PPAR{gamma}-specific agonist (200 nM GW-347845), which displays >103-fold selectively over PPAR{alpha} or PPAR{delta} (for the human and mouse isoforms, –log EC50 values for transactivation are, respectively, 9.2 and 8.9 for PPAR{gamma}, 5.5 and 5.0 for PPAR{alpha}, and 4.6 and 5.0 for PPAR{delta}) (31), B) adenoviral transduction with PPAR{gamma}1, and C) the combination of both treatments (Fig. 3 and Table 2). These three protocols aimed to examine the effects of relatively small, physiological increases in endogenous PPAR{gamma} activity (2- to 10-fold; treatment A), similar to those achieved after the overexpression of active SREBP-1c (Fig. 1) (12) or in the islets of ZDF rats (53), as well as the impact of much larger increases in PPAR{gamma} activity (treatments B and C). By providing much more substantial increases in the transcription of target genes, the latter protocols (B and C) were expected to permit the unambiguous identification of genes whose changes may be too small to detect by microarray analysis when PPAR{gamma} was weakly overexpressed.

Of more than 15,000 endogenous mRNAs examined, the levels of 284 were affected very significantly (P < 0.0099) by infection with the PPAR{gamma}1-encoding adenovirus and incubation with GW-347845 (treatment C). By contrast, 119 and 49 genes, respectively, were altered by expression of PPAR{gamma}1 alone or treatment with GW-347845 after null virus infection (treatments B and A, respectively; Fig. 3). Analysis of clusters (15) revealed three distinct groups of genes that were progressively downregulated [a and b (i)] or upregulated [b (ii) and (iii), c (i) and (ii)] moving through treatments A to C (Fig. 3). Maximal stimulation of PPAR{gamma}1 (treatment C) had no effect on the expression of a number of genes involved in glucose sensing by islet cells, including glucokinase or the ATP-sensitive potassium channel subunits Kir6.2 or SUR1, but did cause a small but significant decrease in mRNA encoding pancreatic duodenum homeobox-1 (PDX-1) (Fig. 3 and Table 2). Similarly, maximal PPAR{gamma}1 activation had no or minor effects on the levels of cell cycle-dependent genes, including cyclin D1, and did not affect the expression of several proapoptotic (Bcl-x, BAD) or antiapoptotic (p53, Bcl2) genes (data not shown and Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. Effects of Ad.PPAR{gamma} and glucose concentration on rat islet gene expression as determined by real-time RT-PCR

 
Of the genes strongly induced in the PPAR{gamma} plus GW-347845-treated group (treatment C), 18 were involved in the {beta}-oxidation of fatty acids by mitochondria (including carnitine-acylcarnitine transferase, acyl-CoA dehydrogenase, {beta}-ketothiolase-acetyl-CoA acyltransferase), peroxisomal fatty acid oxidation (acyl-CoA oxidase), binding (fatty acid-binding protein-4), esterification (diacylglycerol-O-acyltransferase) or transport across the plasma membrane [fatty acid translocase (FAT)/CD36; Table 2]. Of those genes likely to be involved in fatty acid synthesis from glucose, only glycerol phosphate dehydrogenase was significantly induced (Table 2).

Changes in the expression of key genes detected by microarray analysis were quantified by real-time PCR analysis (TaqMan; Table 3). This analysis confirmed that lipogenic genes (acetyl-CoA carboxylase, fatty acid synthase) were minimally affected by PPAR{gamma}1/GW-347845 treatment, whereas GLUT2/Scl2a2 and PDX-1 expression was slightly decreased at 3 and 17 mM glucose, respectively (Table 3). By contrast, RT-PCR analysis revealed that expression of mitochondrial carnitine palmitoyltransferase I and the plasma membrane FAT/CD36 (9) were both strongly induced by PPAR{gamma}1/GW-347845 (Table 3). Conversely, pyruvate dehydrogenase kinase-4 was upregulated in the presence of activated PPAR{gamma}1 (Table 3), a change likely to inhibit mitochondrial pyruvate oxidation (35).

Effects of PPAR{gamma} Activation on Islet Glucose and Fatty Acid Metabolism

As shown in Fig. 4A, the combination of transduction with PPAR{gamma}1 and incubation with GW-347845 (treatment C) significantly diminished islet TG levels. By contrast, the oxidation of exogenously added palmitate was significantly increased by this combined treatment (Fig. 4B), whereas the oxidation of 17 mM glucose was inhibited by >70% under these conditions (Fig. 4C). Correspondingly, glucose-stimulated increases in islet ATP content were completely inhibited by GW-347845 or expression of PPAR{gamma}, in either the presence or absence of the agonist (Table 4), with no associated changes in the expression of mitochondrial uncoupling protein-2 (Table 3). By contrast, culture of nontransfected islets in the presence of the positive agonist alone had no impact on the oxidation of exogenous palmitate or of glucose (Fig. 4, D and E).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4. Effects of Ad.PPAR{gamma}1 and GW-347845 on islet triglyceride (TG) content and palmitate and glucose oxidation. Primary rat islets were treated for 32 h with PPAR{gamma}1 or null adenovirus and then cultured in medium containing 3 mM glucose and 200nM GW-347845 for 16 h (A–C) or treated for 48 h with 200 nM GW-347845 or vehicle before TG extraction (A), [1-14C]palmitate oxidation (B and D), and [U-14C]glucose oxidation (C and E) measurement, as described in MATERIALS AND METHODS. Statistically significant differences between groups: **P < 0.01, ***P < 0.001.

 

View this table:
[in this window]
[in a new window]
 
Table 4. Effect of PPAR{gamma} overexpression on islet ATP content

 
Impact of PPAR{gamma}1 Overexpression on Glucose- and KCl-Stimulated Insulin Secretion

We next determined whether the marked changes in islet glucose metabolism caused by PPAR{gamma} activation were associated with changes in insulin release. As shown in Fig. 5A, glucose-stimulated insulin secretion was significantly reduced after infection of islets with adenoviral PPAR{gamma}1 in either the presence or absence of GW-347845. In contrast, incubation with the PPAR{gamma} agonist alone had no effect on insulin secretion at 17 mM glucose but significantly enhanced release at 3 mM glucose (Fig. 5A). The metabolism-independent stimulation of secretion with 35 mM KCl was unaffected by either PPAR{gamma}1 alone or PPAR{gamma}1 plus GW-347845 (Fig. 5B), consistent with a preserved insulin content and activity of the basal exocytotic machinery under these conditions. Correspondingly, no changes in the levels of mRNAs encoding proteins involved in exocytosis (24), including members of the syntaxin or synaptotagmin families, synaptosomal protein-25 (SNAP25), or Munc18 were revealed by microarray analysis (data not shown), although the expression of Rim1 (Rab3-interacting molecule 1), a known regulator of exocytosis in neurons (24), was decreased (Table 2). Further indicating that the actions of GW-347845 were likely to be mediated by PPAR{gamma}, the effects of the drug on glucose-induced insulin secretion (Fig. 5C) and on the expression of two target genes (Fig. 6) in islets infected with adenoviral PPAR{gamma} were completely reversed with the PPAR{gamma}-selective antagonist GW-9662 (Fig. 5, legend).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Effects of Ad.PPAR{gamma}1 and GW-347845 on glucose- and KCl-stimulated insulin secretion. Islets were treated for 32 h with PPAR{gamma}1 or null adenovirus before overnight (16 h) culture in 3 mM glucose in the presence or absence of 200 nM GW-347845, antagonist (GW-9662), or vehicle. Concentrations of this agent giving IC50 values are 3.3, 7.5, and 32 nM for PPAR{gamma}, -{alpha}, and -{delta}, respectively (28a). Islets were preincubated in 3 mM glucose for 1 h and then incubated in 3 or 17 mM glucose (A and C) or in 3 mM glucose or 3 mM glucose + 35 mM KCl (B) for 30 min before assay of released insulin. Statistically significant differences between groups, calculated as described in MATERIALS AND METHODS: *P < 0.05, **P < 0.01, ***P < 0.001.

 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. PPAR{gamma} antagonist GW-9662 reverses induction of fatty acid translocase (CD36) and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase (ECHDH) mRNA by PPAR{gamma} + GW-347845. Primary rat pancreatic islets were treated for 32 h with Ad.PPAR{gamma} or null adenovirus and then cultured for a further 24 h at 3.0 mM glucose in the presence or absence of 200 nM GW-347845 + GW-9662 at 0.2 or 5 µM as indicated. Levels of mRNAs encoding the indicated genes were analyzed by quantitative real-time RT-PCR. Results are expressed as fold changes over control (null, 3 mM glucose) and presented as means + SE. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001 for effect of PPAR{gamma}.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The principal aim of this study was to identify direct and indirect target genes for PPAR{gamma} in the pancreatic islet. In this way, we sought to assess the potential impact on insulin secretion of overexpression of PPAR{gamma} in certain models of diabetes (53) and the possible effects of long-term therapeutic treatment with PPAR{gamma} agonists.

We demonstrated first that mRNAs encoding both PPAR{gamma}1 and PPAR{gamma}2 isoforms are present in primary rodent islets and that PPAR{gamma}1 and -{gamma}2 levels are considerably increased after the forced expression of SREBP-1c. The latter factor has previously been shown to be upregulated severalfold by glucose in clonal {beta}-cells (2, 51) but more modestly (~60%) in isolated islets (11, 12). Indeed, in the present studies, SREBP-1c expression was not significantly affected by elevated glucose concentrations (Table 3), a difference that may reflect a shorter preincubation period (16 h) at low glucose concentrations than in our previous reports (24 h) (11, 12).

In contrast to the effects of overexpressing activated SREBP-1c in islets (26), increases in PPAR{gamma}1 activity over a broad range had no effect on, or even reduced, the expression of lipogenic genes in the islet (Tables 2 and 3). Moreover, because the expression of key "glucose-sensing" genes (e.g., Kir6.2, SUR1, and glucokinase) was not affected by PPAR{gamma}1 activation, whereas the expression of GLUT2 and PDX-1 was only weakly reduced (Fig. 7, legend), PPAR{gamma} activation alone would appear insufficient to promote the transdifferentiation of islet cells toward an adipocyte phenotype (30, 48). On the contrary, the presence of activated PPAR{gamma}1 decreased islet TG content and glucose oxidation while stimulating fatty acid oxidation (Fig. 4). Thus PPAR{gamma} would seem to play little if any role in mediating the effects of SREBP-1c on gene expression but may, rather, act as a "brake" to oppose the lipogenic effects of the latter factor (see below).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7. Schema showing key sites of action of PPAR{gamma} on glucose and fatty acid metabolism in pancreatic {beta}-cells. Genes whose levels are increased or decreased by activation of PPAR{gamma} (adenovirus + agonist) are indicated by + and –, respectively. Induction of CD36, fatty acyl (FA)-CoA ligase, carnitine palmitoyltransferase I (CPT I), acylcarnitine/carnitine transferase, and enzymes of {beta}-oxidation seem likely to underlie the enhanced oxidation of exogenous palmitate. Conversely, decreases in expression of GLUT2/Slc2a2 and upregulation of pyruvate dehydrogenase kinase (PDK)-1 are likely to reduce glucose oxidation, a change that may be compounded by enhanced oxidation of endogenous FAs and generation of intramitochondrial NADH and acyl-CoA. Note that changes in expression of glucokinase in response to PPAR{gamma} overexpression of activation were not observed in the present study in primary islets, in contrast to recent reports on clonal {beta}-cell lines (26a).

 
Underlying the enhancement of fatty acid oxidation, progressive activation of PPAR{gamma}, achieved by treatment with a specific agonist, overexpression of PPAR{gamma} alone, or the combination of agonist with PPAR{gamma} overexpression, caused a synergistic and coordinated upregulation of key enzymes of mitochondrial and peroxisomal {beta}-oxidation (Fig. 3 and Tables 2 and 3). In addition, changes in the expression of other genes are also likely to contribute to the defective oxidation of glucose and, consequently, glucose-stimulated insulin secretion (Fig. 3 and Table 2). For example, we observed the upregulation of three genes whose elevated levels may disrupt normal glucose oxidation: the plasma membrane lactate transporter MCT (monocarboxylate transporter) 2 (Slc16a7) (1) and aquaporins 3 and 7 (14, 23) (Table 2). Conversely, mitochondrial NADH dehydrogenase subcomplex 5 and the mitochondrial aspartate/glutamate exchanger were progressively suppressed by PPAR{gamma} overexpression and/or activation (Table 2), changes that may decrease the production of mitochondrial coupling factors. Likewise, the expression of connexin 36, involved in the formation of gap junctions and thus cell-cell communication within the islet (6), was also suppressed under these conditions. Finally, we also observed a dramatic increase in the expression of the plasma membrane-associated FAT/CD36 (Tables 2, 3), elevated levels of which may impact on lipid metabolism and, hence, glucose-induced insulin secretion. PPAR{gamma} overexpression/activation had no striking effects on the expression of either pro- or antiapoptotic genes or on the expression of growth factor receptors, consistent with an absence of changes in annexin V binding (L. E. Parton and G. A. Rutter, results not shown).

PPAR{gamma} agonists including troglitazone (32) have previously been reported to activate insulin release acutely, an effect possibly mediated by binding to ATP-sensitive K+ channels (47). A direct action of GW-347845 on these channels may thus contribute to the stimulation of insulin release observed here in response to the agonist alone (Fig. 5A). Although the longer-term effects of PPAR{gamma} activation are consistent with the previously described action of troglitazone to reduce the lipid content of ZDF rat islets (26, 43), the favoring of fatty acid oxidation over synthesis was unexpected. Indeed, in other tissues such a change is more reminiscent of the activation of PPAR{alpha} than PPAR{gamma} (10, 39). Importantly, these effects cannot be ascribed to an upregulation of PPAR{alpha} by overexpressed PPAR{gamma}, since the level of PPAR{alpha} expression was found to be low in control islets and unaffected by any of the treatments used (data not shown). It should be stressed, however, that GW-347845 is expected to be highly selective for the activation of PPAR{gamma} compared with other PPAR isoforms at the concentration used here (see RESULTS). We therefore speculate that the apparent tissue-specific differences in PPAR{gamma} function revealed here may reflect differing levels of PPAR{gamma}-binding partners such as retinoic acid receptors or of coactivators such as CREB-binding protein/p300 or PPAR{gamma} coactivator (PGC)-1 (37). In this context, PGC-1{alpha} was recently shown to be expressed at low levels in nondiabetic islets but induced in two models of diabetes and associated with an inhibition of glucose oxidation and insulin secretion (52). In addition, the complete absence from the islet of adipocyte-specific transcription factors such as CCAAT box enhancer-binding protein-{alpha}/{beta} (8) may limit the ability of PPAR{gamma} to induce the expression of lipid-synthesizing enzymes in islet cells.

Recent studies have indicated that an accumulation of free fatty acids or nonoxidative metabolites, including ceramides (49), rather than the formation of TG (5), may be most closely correlated with apoptotic changes and loss of {beta}-cell function. We show here that the forced activation of PPAR{gamma} in the islet leads to the stimulation of multiple metabolic pathways that favor the disposal of fatty acids (mitochondrial and peroxisomal oxidation, cellular export, and incorporation into diglycerides). Such mechanisms may oppose the deleterious long-term consequences for islet {beta}-cell survival and function of high levels of circulating fatty acids and thus restrict the impact of the glucose-induced upregulation of lipogenic transcription factors including SREBP-1c (2, 51).

A recent report (40) has suggested that PPAR{gamma} in islet {beta}-cells has only a limited role in the antidiabetic effects of TZDs. In the latter studies, the normal expansion of {beta}-cell mass that occurs in control mice in response to high-fat feeding was markedly blunted in PPAR{gamma}-deleted animals. However, PPAR{gamma} deletion in {beta}-cells had no impact on the improvement in glucose tolerance elicited by rosiglitazone in high-fat-fed mice. Whether deletion of PPAR{gamma} in islet {beta}-cells affects the efficacy of TZDs in the context of other models of type 2 diabetes remains to be established.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by a Biological and Biotechnology Research Council Studentship to L. E. Parton, grants to G. A. Rutter from the Medical Research Council (UK) and the Wellcome Trust (Programme Grant no. 067081/Z/02/Z), and the Marie Curie Fellowships Association (to F. Diraison). G. A. Rutter is a Wellcome Trust Research Leave Fellow.


    ACKNOWLEDGMENTS
 
We thank John L. Andrews, Robert Mertz, Michelle M. Eilert, Rebecca Rowe, and Debbie Martin for technical help.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. A. Rutter, Henry Wellcome Signalling Laboratories and Dept. of Biochemistry, School of Medical Sciences, University Walk, Univ. of Bristol, Bristol, BS8 1TD UK (E-mail: g.a.rutter{at}bris.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ainscow EK, Zhao C, and Rutter GA. Acute overexpression of lactate dehydrogenase-A perturbs beta-cell mitochondrial metabolism and insulin secretion. Diabetes 49: 1149–1155, 2000.[Abstract]
  2. Andreolas C, da Silva Xavier G, Diraison F, Zhao C, Varadi A, Lopez-Casillas F, Ferre P, Foufelle F, and Rutter GA. Stimulation of acetyl-CoA carboxylase gene expression by glucose requires insulin release and sterol regulatory element binding protein 1c in pancreatic MIN6 beta-cells. Diabetes 51: 2536–2545, 2002.[Abstract/Free Full Text]
  3. Bollheimer LC, Troll S, Landauer H, Wrede CE, Scholmerich J, and Buettner R. Insulin-sparing effects of troglitazone in rat pancreatic islets. J Mol Endocrinol 31: 61–69, 2003.[Abstract/Free Full Text]
  4. Braissant O, Foufelle F, Scotto C, Dauca M, and Wahli W. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology 137: 354–366, 1996.[Abstract]
  5. Briaud I, Harmon JS, Kelpe CL, Segu VB, and Poitout V. Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes 50: 315–321, 2001.[Abstract/Free Full Text]
  6. Calabrese A, Guldenagel M, Charollais A, Mas C, Caton D, Bauquis J, Serre-Beinier V, Caille D, Sohl G, Teubner B, Le Gurun S, Trovato-Salinaro A, Condorelli DF, Haefliger JA, Willecke K, and Meda P. Cx36 and the function of endocrine pancreas. Cell Commun Adhes 8: 387–391, 2001.[ISI][Medline]
  7. Cavaghan MK, Ehrmann DA, Byrne MM, and Polonsky KS. Treatment with the oral antidiabetic agent troglitazone improves beta cell responses to glucose in subjects with impaired glucose tolerance. J Clin Invest 100: 530–537, 1997.[Abstract/Free Full Text]
  8. Christy RJ, Yang VW, Ntambi JM, Geiman DE, Landschulz WH, Friedman AD, Nakabeppu Y, Kelly TJ, and Lane MD. Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes. Genes Dev 3: 1323–1335, 1989.[Abstract]
  9. Coburn CT, Hajri T, Ibrahimi A, and Abumrad NA. Role of CD36 in membrane transport and utilization of long-chain fatty acids by different tissues. J Mol Neurosci 16: 117–121, 151–117, 2001.[ISI][Medline]
  10. Desvergne B and Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20: 649–688, 1999.[Abstract/Free Full Text]
  11. Diraison F, Ferre P, Foufelle F, and Rutter GA. Impact of over-expression of sterol response element binding protein-1c (SREBP-1c) on glucose-stimulated insulin secretion from rat islets (Abstract). Diabetologia 45: 460, 2002.
  12. Diraison F, Parton L, Ferre P, Foufelle F, Briscoe CP, Leclerc I, and Rutter GA. Over-expression of sterol regulatory element binding protein-1c in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside. Biochem J 373: 769–778, 2004.
  13. Dubois M, Pattou F, Kerr-Conte J, Gmyr V, Vandewalle B, Desreumaux P, Auwerx J, Schoonjans K, and Lefebvre J. Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in normal human pancreatic islet cells. Diabetologia 43: 1165–1169, 2000.[CrossRef][ISI][Medline]
  14. Echevarria M, Windhager EE, Tate SS, and Frindt G. Cloning and expression of AQP3, a water channel from the medullary collecting duct of rat kidney. Proc Natl Acad Sci USA 91: 10997–11001, 1994.[Abstract/Free Full Text]
  15. Eisen MB, Spellman PT, Brown PO, and Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95: 14863–14868, 1998.[Abstract/Free Full Text]
  16. Elbrecht A, Chen Y, Cullinan CA, Hayes N, Leibowitz M, Moller DE, and Berger J. Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gamma 1 and gamma 2. Biochem Biophys Res Commun 224: 431–437, 1996.[CrossRef][ISI][Medline]
  17. Fajas L, Fruchart JC, and Auwerx J. PPARgamma3 mRNA: a distinct PPARgamma mRNA subtype transcribed from an independent promoter. FEBS Lett 438: 55–60, 1998.[CrossRef][ISI][Medline]
  18. Foretz M, Pacot C, Dugail I, Lemarchand P, Guichard C, Le Liepvre X, Berthelier-Lubrano C, Spiegelman B, Kim JB, Ferre P, and Foufelle F. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose. Mol Cell Biol 19: 3760–3768, 1999.[Abstract/Free Full Text]
  19. Gotoh M, Maki T, Satomi S, Porter J, Bonner-Weir S, O'Hara CJ, and Monaco AP. Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase injection. Transplantation 43: 725–730, 1987.[ISI][Medline]
  20. Gurnell M, Savage DB, Chatterjee VK, and O'Rahilly S. The metabolic syndrome: peroxisome proliferator-activated receptor gamma and its therapeutic modulation. J Clin Endocrinol Metab 88: 2412–2421, 2003.[Abstract/Free Full Text]
  21. Hara K, Okada T, Tobe K, Yasuda K, Mori Y, Kadowaki H, Hagura R, Akanuma Y, Kimura S, Ito C, and Kadowaki T. The Pro12Ala polymorphism in PPAR gamma2 may confer resistance to type 2 diabetes. Biochem Biophys Res Commun 271: 212–216, 2000.[CrossRef][ISI][Medline]
  22. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, and Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95: 2509–2514, 1998.[Abstract/Free Full Text]
  23. Ishibashi K, Kuwahara M, Gu Y, Kageyama Y, Tohsaka A, Suzuki F, Marumo F, and Sasaki S. Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J Biol Chem 272: 20782–20786, 1997.[Abstract/Free Full Text]
  24. Jahn R, Lang T, and Sudhof TC. Membrane fusion. Cell 112: 519–533, 2003.[ISI][Medline]
  25. Johnson JH, Ogawa A, Chen L, Orci L, Newgard CB, Alam T, and Unger RH. Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes. Science 250: 546–549, 1990.[ISI][Medline]
  26. Kakuma T, Lee Y, Higa M, Wang Z, Pan W, Shimomura I, and Unger RH. Leptin, troglitazone, and the expression of sterol regulatory element binding proteins in liver and pancreatic islets. Proc Natl Acad Sci USA 97: 8536–8541, 2000.[Abstract/Free Full Text]
  27. Kim H, Cha J-Y, Kim S-Y, Kim J, Roh KJ, Seong J-K, Lee NT, Choi KY, Kim K-S, and Ahn Y-H. Peroxisome proliferator-activated receptor-{gamma} upregulates glucokinase gene expression in {beta}-cells. Diabetes 51: 676–685, 2002.[Abstract/Free Full Text]
  28. Kubota N, Terauchi Y, Miki H, Tamemoto H, Yamauchi T, Komeda K, Satoh S, Nakano R, Ishii C, Sugiyama T, Eto K, Tsubamoto Y, Okuno A, Murakami K, Sekihara H, Hasegawa G, Naito M, Toyoshima Y, Tanaka S, Shiota K, Kitamura T, Fujita T, Ezaki O, Aizawa S, Nagai R, Tobe K, Kimura S, and Kadowaki T. PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell 4: 597–609, 1999.[ISI][Medline]
  29. Lee Y, Hirose H, Ohneda M, Johnson JH, McGarry JD, and Unger RH. Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships. Proc Natl Acad Sci USA 91: 10878–10882, 1994.[Abstract/Free Full Text]
  30. Leesnitzer LM, Parks DJ, Bledsoe RK, Cobb JE, Collins JL, Consler TG, Davis RG, Hull-Ryde EA, Lenhard JM, Patel L, Plunket KD, Shenk JL, Stimmel JB, Therapontos C, Willson TM, and Blanchard SG. Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry 41: 6640–6650, 2002.[CrossRef][ISI][Medline]
  31. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, and Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem 270: 12953–12956, 1995.[Abstract/Free Full Text]
  32. Li Y and Lazar MA. Differential gene regulation by PPARgamma agonist and constitutively active PPARgamma2. Mol Endocrinol 16: 1040–1048, 2002.[Abstract/Free Full Text]
  33. Liu KG, Lambert MH, Ayscue AH, Henke BR, Leesnitzer LM, Oliver WR Jr, Plunket KD, Xu HE, Sternbach DD, and Willson TM. Synthesis and biological activity of L-tyrosine-based PPARgamma agonists with reduced molecular weight. Bioorg Med Chem 11: 3111–3113, 2001.[CrossRef]
  34. Masuda K, Okamoto Y, Tsuura Y, Kato S, Miura T, Tsuda K, Horikoshi H, Ishida H, and Seino Y. Effects of troglitazone (CS-045) on insulin secretion in isolated rat pancreatic islets and HIT cells: an insulinotropic mechanism distinct from glibenclamide. Diabetologia 38: 24–30, 1995.[CrossRef][ISI][Medline]
  35. Mori H, Ikegami H, Kawaguchi Y, Seino S, Yokoi N, Takeda J, Inoue I, Seino Y, Yasuda K, Hanafusa T, Yamagata K, Awata T, Kadowaki T, Hara K, Yamada N, Gotoda T, Iwasaki N, Iwamoto Y, Sanke T, Nanjo K, Oka Y, Matsutani A, Maeda E, and Kasuga M. The Pro12->Ala substitution in PPAR-gamma is associated with resistance to development of diabetes in the general population: possible involvement in impairment of insulin secretion in individuals with type 2 diabetes. Diabetes 50: 891–894, 2001.[Abstract/Free Full Text]
  36. Nakamichi Y, Kikuta T, Ito E, Ohara-Imaizumi M, Nishiwaki C, Ishida H, and Nagamatsu S. PPAR-gamma overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells. Biochem Biophys Res Commun 306: 832–836, 2003.[CrossRef][ISI][Medline]
  37. Nicholls LI, Ainscow EK, and Rutter GA. Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets. Biochem Biophys Res Commun 291: 1081–1088, 2002.[CrossRef][ISI][Medline]
  38. Prentki M and Corkey BE. Are the beta-cell signaling molecules malonyl-CoA and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM? Diabetes 45: 273–283, 1996.[Abstract]
  39. Puigserver P, Wu Z, Park CW, Graves R, Wright M, and Spiegelman BM. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92: 829–839, 1998.[ISI][Medline]
  40. Ristow M, Muller-Wieland D, Pfeiffer A, Krone W, and Kahn CR. Obesity associated with a mutation in a genetic regulator of adipocyte differentiation. N Engl J Med 339: 953–959, 1998.[Abstract/Free Full Text]
  41. Roduit R, Morin J, Masse F, Segall L, Roche E, Newgard CB, Assimacopoulos-Jeannet F, and Prentki M. Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta-cell. J Biol Chem 275: 35799–35806, 2000.[Abstract/Free Full Text]
  42. Rosen ED, Kulkarni RN, Sarraf P, Ozcan U, Okada T, Hsu CH, Eisenman D, Magnuson MA, Gonzalez FJ, Kahn CR, and Spiegelman BM. Targeted elimination of peroxisome proliferator-activated receptor gamma in beta cells leads to abnormalities in islet mass without compromising glucose homeostasis. Mol Cell Biol 23: 7222–7229, 2003.[Abstract/Free Full Text]
  43. Savage DB, Tan GD, Acerini CL, Jebb SA, Agostini M, Gurnell M, Williams RL, Umpleby AM, Thomas EL, Bell JD, Dixon AK, Dunne F, Boiani R, Cinti S, Vidal-Puig A, Karpe F, Chatterjee VK, and O'Rahilly S. Human metabolic syndrome resulting from dominant-negative mutations in the nuclear receptor peroxisome proliferator-activated receptor-gamma. Diabetes 52: 910–917, 2003.[Abstract/Free Full Text]
  44. Shimabukuro M, Ohneda M, Lee Y, and Unger RH. Role of nitric oxide in obesity-induced beta cell disease. J Clin Invest 100: 290–295, 1997.[Abstract/Free Full Text]
  45. Shimabukuro M, Zhou YT, Lee Y, and Unger RH. Troglitazone lowers islet fat and restores beta cell function of Zucker diabetic fatty rats. J Biol Chem 273: 3547–3550, 1998.[Abstract/Free Full Text]
  46. Shimabukuro M, Zhou YT, Levi M, and Unger RH. Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci USA 95: 2498–2502, 1998.[Abstract/Free Full Text]
  47. Spiegelman BM and Flier JS. Adipogenesis and obesity: rounding out the big picture. Cell 87: 377–389, 1996.[ISI][Medline]
  48. Storey JD and Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad Sci USA 100: 9440–9445, 2003.[Abstract/Free Full Text]
  49. Sunaga Y, Inagaki N, Gonoi T, Yamada Y, Ishida H, Seino Y, and Seino S. Troglitazone but not pioglitazone affects ATP-sensitive K(+) channel activity. Eur J Pharmacol 381: 71–76, 1999.[CrossRef][ISI][Medline]
  50. Tontonoz P, Hu E, Graves RA, Budavari AI, and Spiegelman BM. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8: 1224–1234, 1994.[Abstract]
  51. Unger RH and Zhou YT. Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover. Diabetes 50, Suppl 1: S118–S121, 2001.
  52. Varadi A, Molnar E, Ostenson CG, and Ashcroft SJ. Isoforms of endoplasmic reticulum Ca(2+)-ATPase are differentially expressed in normal and diabetic islets of Langerhans. Biochem J 319: 521–527, 1996.[ISI][Medline]
  53. Wang H, Maechler P, Antinozzi PA, Herrero L, Hagenfeldt-Johansson KA, Bjorklund A, and Wollheim CB. The transcription factor SREBP-1c is instrumental in the development of beta-cell dysfunction. J Biol Chem 278: 16622–16629, 2003.[Abstract/Free Full Text]
  54. Yoon JC, Xu G, Deeney JT, Yang SN, Rhee J, Puigserver P, Levens AR, Yang R, Zhang CY, Lowell BB, Berggren PO, Newgard CB, Bonner-Weir S, Weir G, and Spiegelman BM. Suppression of beta cell energy metabolism and insulin release by PGC-1alpha. Dev Cell 5: 73–83, 2003.[ISI][Medline]
  55. Zhou Y, Shimabukuro M, Wang M, Lee Y, Higa M, Milburn JL, Newgard C, and Unger RH. Role of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells. Proc Natl Acad Sci USA 95: 8898–8903, 1998.[Abstract/Free Full Text]