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Am J Physiol Endocrinol Metab 291: E982-E994, 2006. First published June 13, 2006; doi:10.1152/ajpendo.00067.2006
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Limited role for SREBP-1c in defective glucose-induced insulin secretion from Zucker diabetic fatty rat islets: a functional and gene profiling analysis

Laura E. Parton,1 Patrick J. McMillen,2 Yingnian Shen,2 Elizabeth Docherty,2 Erin Sharpe,2 Frédérique Diraison,1 Celia P. Briscoe,3 and Guy A. Rutter1

1Henry Wellcome Signaling Laboratories and Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom; and Departments of 2Disease and Biotranscriptomics and 3Metabolic Diseases, GlaxoSmithKline, Research Triangle Park, North Carolina

Submitted 8 February 2006 ; accepted in final form 26 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Accumulation of intracellular lipid may contribute to defective insulin secretion in type 2 diabetes. Although Zucker diabetic fatty (ZDF; fa/fa) rat islets are fat-laden and overexpress the lipogenic master gene, sterol regulatory element binding protein 1c (SREBP-1c), the contribution of SREBP-1c to the secretory defects observed in this model remains unclear. Here we compare the gene expression profile of lean control (fa/+) and ZDF rat islets in the absence or presence of dominant-negative SREBP-1c (SREBP-1c DN). ZDF islets displayed elevated basal insulin secretion at 3 mmol/l glucose but a severely depressed response to 17 mmol/l glucose. While SREBP-1c DN reduced basal insulin secretion from ZDF islets, glucose-stimulated insulin secretion was not improved. Of 57 genes differentially regulated in ZDF islets and implicated in glucose metabolism, vesicle trafficking, ion fluxes, and/or exocytosis, 21 were upregulated and 5 were suppressed by SREBP-1c DN. Genes underrepresented in ZDF islets were either unaffected (Glut-2, Kir6.2, Rab3), stimulated (voltage-dependent Ca2+ channel subunit {alpha}1D, CPT2, SUR2, rab9, syt13), or inhibited (syntaxin 7, secretogranin-2) by SREBP-1c inhibition. Correspondingly, SREBP-1c DN largely corrected decreases in the expression of the transcription factors Pdx-1 and MafA but did not affect the abnormalities in Pax6, Arx, hepatic nuclear factor-1{alpha} (HNF1{alpha}), HNF3beta/Forkhead box-a2 (Foxa2), inducible cyclic AMP early repressor (ICER), or transcription factor 7-like 2 (TCF7L2) expression observed in ZDF islets. We conclude that upregulation of SREBP-1c and mild increases in triglyceride content do not explain defective glucose-stimulated insulin secretion from ZDF rats. However, overexpression of SREBP-1c may contribute to enhanced basal insulin secretion in this model.

pancreatic islets; sterol regulatory element binding protein-1c; glucolipotoxicity

GLUCOSE STIMULATES THE SECRETION of insulin from the pancreatic islet beta-cell by increasingly well-understood mechanisms (52). These involve uptake of glucose by a low-affinity glucose transporter GLUT2 (in rodents), enhanced mitochondrial ATP synthesis, and closure of ATP-sensitive K+ channels (KATP channels). Opening of voltage-gated Ca2+ channels and influx of Ca2+ ions then lead to the movement of dense core secretory granules (LDCVs) toward the plasma membrane and exocytosis.

Although the generation of lipid signaling molecules, including malonyl-CoA and long chain acyl-CoAs, has also been proposed to play an important role in glucose-stimulated insulin secretion (GSIS) in the short term (14, 49), accumulation of intracellular lipids in the longer term (48, 67) has been proposed to contribute to both defective glucose signaling (16) and diminished beta-cell mass (8) in type 2 diabetes (T2D). An example of the latter is provided by the Zucker diabetic fatty (ZDF) rat. In this established model of T2D associated with homozygous deletion of the leptin receptor fa and overfeeding, time-dependent triglyceride accumulation in islet cells (38), decreased beta-cell glucose transporter-2 (GLUT2, slc2a2) expression (35), increased beta-cell apoptosis (57), elevated basal insulin secretion, and loss of GSIS (62) are all observed.

Sterol regulatory element binding protein-1c (SREBP-1c) is a master regulator of lipogenic gene expression (58, 63), which is normally expressed at very low levels in islet beta-cells. Although deletion of the SREBP-1 gene in mice has only minor effects on insulin secretion (61), enhanced expression of this factor may play a role in beta-cell failure. Thus SREBP-1c levels are increased in beta-cells in response to elevated glucose concentrations (4, 18, 19, 53) and in islets from both ZDF (11) and diet-induced obese (36) rats. Furthermore, ectopic overexpression of a constitutively active form of SREBP-1c results in triglyceride accumulation and suppressed GSIS in clonal beta-cells (4, 70, 76) and rat islets (18, 61). Interestingly, chronic exposure to high glucose concentrations or overexpression of activated SREBP-1c induced an endoplasmic reticulum stress response in tumoral INS-1 beta-cells, and the effects of the former were blocked by SREBP-1c inactivation (69).

To analyze the role of more physiological changes in SREBP-1c levels in the primary beta-cell, we have determined here the effects of inactivating SREBP-1c in islets isolated from control and ZDF rats. We show that, despite contributing to the dysregulated expression of a large number of genes in ZDF islets, elevated SREBP-1c levels and lipid accumulation do not alone explain defective GSIS in this model. On the other hand, the results of SREBP-1c inactivation suggest that this factor plays an important role in elevated basal insulin release in ZDF rats. Differences between the gene profiles of ZDF rat islets vs. human T2D islets (23), and their respective controls, are also highlighted, and the potential role of new candidate genes involved in the loss of GSIS in ZDF islets is discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Obese inbred male ZDF rats (ZDF/Gmi-fa/fa) and heterozygous (ZDF/Gmi-fa/+) lean littermates were purchased from Charles River Laboratories (Wilmington, MA) at 6 wk of age and housed for 1 wk before use. All animal procedures were carried out in accordance with UK Home Office welfare guidelines and project license restrictions.

Genotyping. Total DNA was extracted from tail snips by proteinase K digestion. A fragment of Ob-Rb was amplified by PCR as described previously (47). Primers (5'-GTT TGC GTA TGG AAG TCA CAG-3' and 5'-ACC AGC AGA GAT GTA TCC GAG-3') were used to amplify products from 100 ng of genomic DNA which were then digested with Msp1 and analyzed on 1% (wt/vol) agarose gels.

Plasma metabolite and hormone measurements. Cardiac blood samples (0.5 ml) were collected from fed rats immediately after death and placed in heparinized tubes on ice. Plasma was separated by centrifugation within 30 min and frozen at –80°C until assay. Measurements of plasma glucose and triglyceride were performed using an Olympus Au640 clinical chemistry analyzer (Olympus America) at 37°C, using hexokinase and lipase-glucokinase (GK), respectively. Plasma insulin was measured by radioimmunoassay (Linco Research, St. Charles, MO).

Islet isolation and culture. Pancreatic islets were isolated from 7-wk-old ZDF rats by perfusion of the pancreatic duct and in situ collagenase digestion (Boehringer Mannheim, Mannheim, Germany) as described (21). Islets were subsequently purified on Histopaque gradient solutions (10 ml of 1.119 g/l, 6 ml of 1.083 g/l, 6 ml of 1.077 g/l; Sigma, Poole, Dorset, UK). Isolated islets were cultured in suspension for 16 h in RPMI-1640 containing 10% (vol/vol) FCS, 10 mmol/l glucose, 2 mmol/l glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin and incubated at 37°C with 95% air-5% CO2. Islets were handpicked 24 h later.

Amplification of adenoviruses. Adenoviruses encoding dominant-negative SREBP-1c (SREBP-1c DN; amino acids 1–403, Y-320 -> R) or enhanced green fluorescent protein (eGFP; "null" virus) were constructed as described previously (20, 26, 37) and used at a multiplicity of infection (MOI) of 50 viral particles/cell. Viruses were amplified in HEK293 cells and purified on a CsCl gradient (2). Islets were cultured for 16 h after isolation in RPMI medium as supplemented above and infected with adenoviruses at an MOI of 50 viral particles/cell for a further 72 h before use.

Oil red-O lipid staining. Islets were washed with PBS and fixed with Zamboni's fixative (59) overnight at 4°C for preparation of islet cryostat sections (46).

Microarray studies: sample preparation and processing. Total RNA from four separate islet cultures were isolated by TRI reagent and purified on an RNeasy column (Qiagen, Valencia, CA) before labeling for hybridization to Affymetrix RAE230Plus rat arrays (Affymetrix, Santa Clara, CA). Total RNA was amplified and labeled with the Nugen (San Carlos, CA) Ovation system following the kit's standard protocol (http://www.nugentechnologies.com/pdfs/Ovation_Biotin_UserGuide.pdf). Briefly, each sample was processed from 50 ng of total RNA that were first converted to double-stranded cDNA with a short RNA sequence incorporated into one of the strands to allow for an amplification process ("SPIA") in the next step. Using all the material created from the double-stranded cDNA reaction, we performed Ribo-SPIA linear amplification. The SPIA DNA product was purified, fragmented, and labeled; 2.2 µg of purified and labeled DNA were used in the hybridization cocktail, and the chips were hybridized following the Affymetrix recommendations (GeneChip Expression Analysis Technical Manual, http://www.affymetrix.com/support/technical/manual/expression_manual.affx; 2003). Hybridized chips were washed and then scanned on an Affymetrix GeneChip 3000 confocal scanner. Gene expression data were generated by GeneChip Operating Software (GCOS) from Affymetrix. Total RNA and double-stranded cDNA SPIA product were checked for quality (RNA 6000 Nano LabChip with the Bioanalyzer system; Agilent Technologies, Palo Alto, CA).

Microarray studies: data analysis. From the GCOS, we obtained an expression signal and a Present/Absent call status for every probe set on each chip. All expression data were normalized by global scaling to a trimmed average intensity of 150 per chip. The Present/Absent call status was not used as a criterion for gene selection. All gene signals were logged (to base 2) after the addition of 1 intensity unit to all values so as to eliminate the presence of any negative log values. Using all gene signals as variables, correlation coefficients were calculated within each group of four replicates for each treatment group. Using median absolute deviation scores derived from the coefficients and principle component analysis on the first two components, we identified outliers: one sample from each of the four treatment groups was eliminated from the rest of the analysis, giving three samples per group with four groups total.

The mean signal for all genes within replicate samples was calculated on the logged signals, and log ratios of the average signals were calculated. Statistical significance of changes was assessed by homoscedastic two-tailed Student's t-test for unpaired samples, and a false discovery rate was calculated using the Q-value program (R statistical package). Further gene filtering was handled by use of a t-test P value threshold of 0.01 (unless otherwise indicated) and a Q-value threshold of 0.1. Additionally, an intensity threshold was used to decrease the number of genes that were significant because of the bias at low signal ranges. Only genes for which one of the comparison groups had a mean gene intensity of 50 units (5.6439 in log 2) or more were continued further.

Real-time quantitative RT-PCR. Total islet RNA was extracted using TRIzol (Invitrogen, Paisley, UK) 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). Complementary DNA was synthesized from 1 µg of total RNA using random hexamers and murine moloney leukemia virus RT (Applied Biosystems). All genes, primers, and probes used in real-time RT-PCR analysis were obtained from Biosource International (Camarillo, CA) and are described previously (46), with the exception of adipocyte protein-2{gamma} (AP-2{gamma}: forward 5'-CTGAGAACCTAGGGCTGCAC-3', reverse 5'-GGTCCTTTGCGAATGA CAGT-3'; probe: 5'-FAM-CCCGCATCTGCTCCTACACGAT-3'-TAMRA). PCR was performed using 25 ng of reverse-transcribed total RNA with 300 nM sense and anti-sense primers, 100 nM dual-labeled probe (5'-FAM, 3'-TAMRA), 4.0 mmol/l MgCl2, 300 mmol/l deoxyribonucleotide triphosphate (dNTP), and 1.25 units of HotStar Taq polymerase (Qiagen, Crawley, UK) in a total volume of 25 µl in a DNA Engine Opticon-2 Real-Time PCR Detection system (MJ Research, Waltham, MA). Standard curves were constructed by amplifying serial dilutions of untreated rat islet cDNA (50 ng to 0.64 pg) and plotting cycle threshold (CT) values as a function of starting reverse-transcribed RNA. mRNA expression of target genes was normalized to the housekeeping gene cyclophilin A.

Microarray data have been submitted for deposition at the minimum information about a microarray experiment (MIAME) database (accession nos. awaited).

Intracellular triglyceride measurements. Total lipids were extracted from 150 rat islets using a chloroform-methanol (2:1, vol/vol) mixture, and triglyceride (TG) was quantified as described (Infinity TG Reagent, Sigma) (18).

Measurements of insulin secretion and total insulin content. Islets were incubated for 30 min at 37°C in 2 ml of Krebs-Ringer bicarbonate HEPES buffer (KRHB; 130 mmol/l NaCl, 3.6 mmol/l KCl, 1.5 mmol/l CaCl2, 0.5 mmol/l MgSO4, 0.5 mmol/l KH2PO4, 2.0 mmol/l NaHCO3, and 10 mmol/l HEPES) supplemented with 3 mmol/l glucose and 0.2% (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 for 1 h in 1 ml of KRHB, as above, containing 3 or 17 mmol/l glucose. Total insulin was extracted in acidified ethanol (75% ethanol, 23.5% H2O, and 1.5% HCl) with sonication. Insulin was measured by radioimmunoassay, as given above.

Intracellular free Ca2+ concentration measurements. Islets were dissociated into small clusters and single cells by gentle pipetting in Ca2+-free medium based on Hanks' balanced salt solution (Invitrogen) as described previously (50). Clusters and single cells were cultured for 1 day on circular 24-mm glass coverslips. Cells were loaded with 2 µM fura 2-acetoxymethylester (Sigma) for 30 min at 37°C in KRHB containing 3 mmol/l glucose. Cells were then transferred into a thermostatically controlled chamber at 37°C, mounted on the stage of an inverted microscope in the epifluorescence mode with a x40 objective (UAPO/340 40x/1.35, Olympus). Excitation was alternately at 340 and 380 nm (0.2 Hz), using a monochromator (polychrome IV, Tillphotonics). Emission signals were detected at 515 nm with a cooled charge-coupled device camera. Intracellular free Ca2+ concentration ([Ca2+]) is expressed as the ratio of fluorescence at 340/380 nm.

Total internal reflection of fluorescence imaging and calculation of diffusion coefficients. Dissociated isolated cells were plated onto untreated coverslips (n = 1.53). After 6 h, cells were infected (MOI 50–100) with adenovirus encoding neuropeptide Y-Venus (NPY.Venus) (66) and cultured for a further 24–48 h before imaging experiments. A total internal reflection of fluorescence (TIRF) microscope fitted with a numerical aperture 1.45 (PlanAPO 100x/1.45 TIRFM, Olympus) objective lens was used for imaging (excitation 488 nm, emission 515 nm) during perifusion of cells in KRHB medium (see above) initially containing 3 mmol/l glucose. Diffusion coefficients for individual dense core vesicles were determined as described previously (64). Exocytotic events were scored as those in which vesicles rapidly brightened (because of the increased fluorescence of NPY.Venus prompted on vesicle fusion at the plasma membrane and luminal alkalanization) and then disappeared rapidly, usually with the formation of a "fluorescence cloud" of released fluorophore (66).

Statistical analysis. Functional analyses were performed at least three times in triplicate, and data are presented as means ± SE or means ± SD as stated. Statistical significance was assessed by the Student's t-test for unpaired comparison and two-tailed analysis or by ANOVA followed by a Newman-Keuls test, as appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General characteristics of ZDF animals and islets. Islets were isolated from heterozygous lean (fa/+) or homozygous obese inbred (Gmi fa/fa) male ZDF rats at 7 wk of age. Body weight, blood glucose, plasma insulin, and TG levels are shown in Table 1. At the time of islet isolation, ZDF animals were obese, mildly hyperglycemic, and severely hyperinsulinemic. Glucose (17 vs. 3 mmol/l)-stimulated insulin secretion was significantly suppressed, from 11-fold in islets from fa/+ animals to ~2-fold in ZDF fa/fa islets, whereas basal insulin secretion was elevated by 2.5-fold in the latter (see below, GoGoGoGoFig. 5A).


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  		Table 1. Plasma metabolite and hormone levels in 7-wk-old ZDF rats

 

Figure 1
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  		Fig. 1. Glucose and potassium-induced intracellular free Ca2+ concentration ([Ca2+]i) changes in islet clusters from Zucker diabetic fatty (ZDF) rats. Fura 2-AM-loaded islets were perifused with Krebs-Ringer bicarbonate HEPES buffer (KRHB) containing 3 mmol/l glucose (G3.0) for 5 min before stimulation with KRHB containing either 17 mmol/l glucose (G17) or 30 mmol/l KCl (K30) for an additional 15 min. A and C, B and D: representative traces for lean (fa/+) and ZDF (fa/fa) islets, respectively. B: traces i (gray) and ii (black) show 2 typical responses to high glucose of cells from an fa/fa ZDF rat islet. E and F: mean traces for 16–20 islet clusters from 3 fa/+ (black) or fa/fa (gray) ZDF rats. G: mean values for area under the curve for the period 0–7 min after the addition of glucose or a high concentration (30 mmol/l) of KCl.

 

Figure 2
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  		Fig. 2. Increased sterol regulatory element binding protein 1c (SREBP-1c) mRNA expression and lipid content in ZDF islets. Isolated islets from ZDF or lean control rats were cultured with dominant-negative SREBP-1c (SREBP DN) or null adenoviruses for 72 h. A: relative SREBP-1c mRNA expression as analyzed by quantitative real-time RT-PCR. B: total islet triglyceride (TG) content (ng/islet). Results are presented as means ± SE of 4 independent experiments. C: representative oil red-O images of islet cryostat sections of lean (i and ii) and ZDF (iii and iv) islets infected with null and SREBP DN adenoviruses, respectively.

 

Figure 3
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  		Fig. 3. Inhibition of endogenous SREBP-1c reduces lipogenic gene expression in ZDF islets. Islets isolated from ZDF or lean control rats were cultured with SREBP DN or null adenoviruses for 72 h before extraction of total RNA. mRNA levels of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}; A), adipocyte protein-2{gamma} (AP-2{gamma}) transcription factor (B), diacylglycerol acyltransferase-2 (DGAT-2; C), and GLUT-2 (D) were analyzed by quantitative real-time RT-PCR. Results are expressed as fold change over control (lean fa/+) and presented as means ± SE of 4 independent experiments.

 

Figure 4
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  		Fig. 4. Differential expression of genes in ZDF vs. fa/+ islets as determined by microarray analysis. RNA was isolated from pooled islet preparations from 3 lean or ZDF animals in 4 independent experiments. Individual cRNA preparations were hybridized to Affymetrix U230 chips. Graphs show changes in mRNAs encoding the following. A: pancreatic hormones. B: lipogenic enzymes. C: glucose sensors. D: cytoskeleton remodeling and vesicle transport. E: exocytosis and vesicle maturation. (For definitions of abbreviations, see RESULTS.) Results are expressed as fold change over control (lean fa/+) and presented as means ± SE. **P < 0.01, ***P < 0.001.

 

Figure 5
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  		Fig. 5. Inhibition of endogenous SREBP-1c rescues basal, but not glucose-stimulated, insulin secretion in ZDF islets. A: islets isolated from ZDF or lean control rats were cultured with SREBP DN or null adenoviruses for 72 h before assay of released insulin. Islets were preincubated in KRHB containing 3 mmol/l glucose for 30 min before stimulation with 17 mmol/l glucose for 1 h. Results are expressed as a percentage of total islet insulin content, presented as means ± SE of 4 independent experiments. B: measurements of total insulin content of islets, as above.

 
To determine whether the observed changes in insulin release might reflect alterations in the dynamics of insulin-containing vesicles in single cells, we used TIRF microscopy analysis. The number of plasma membrane-docked vesicles revealed using the expressed recombinant fluorescent insulin surrogate, NPY.Venus (66), was unaltered in fa/+ (14.65 ± 0.78 vesicles/10 µm2) vs. ZDF (14.26 ± 0.84 per 10 µm2) islet cells. However, in response to elevated glucose concentrations (17 vs. 3 mmol/l), exocytosis of individual NPY.Venus-containing vesicles was frequently detected in fa/+ islet cells (4/12 cells, 4 preparations) but was not apparent in ZDF islet-derived cells (0/12 cells). Although falling below statistical significance, calculated vesicle diffusion coefficients tended to be slightly augmented at low glucose concentrations in ZDF vs. control islet cells, but unaltered or decreased in response to high glucose or KCl in ZDF islet-derived cells (data not shown).

To assess whether the above changes might be due to alterations in intracellular free [Ca2+] ([Ca2+]i), we next used the trappable intracellular dye fura 2 and microfluorimetry. As shown in Fig. 1 (A vs. B, C vs. D), basal [Ca2+]i was essentially identical in ZDF islet and fa/+ (lean) islet-derived cells. However, whereas control cells responded to 17 mmol/l glucose with clear oscillatory increases in [Ca2+]i, the majority (9/12 cells, 4 preparations) of ZDF islet-derived cells failed to display any significant increase in [Ca2+]i (Fig. 1Bi) or only a significantly delayed (~4 vs. 1 min to onset) increase in [Ca2+]i (Fig. 1Bii) in response to high glucose. By contrast, depolarization with 30 mmol/l KCl elicited an identical increase in [Ca2+]i in fa/+ and ZDF islets (Fig. 1, C vs. D). Mean traces for >12 cells are shown for each stimulus in Fig. 1, E and F, and the integrated increases in [Ca2+]i ("area under curve") in Fig. 1G.

Expression of SREBP-1c DN decreases TG content of ZDF islets and partially rescues abnormal gene expression. SREBP-1c mRNA was overexpressed (1.7-fold increase, P < 0.001) in ZDF (fa/fa) compared with lean control (fa/+) islets (Fig. 2A), consistent with previous results (36). As expected, islet TG content was substantially elevated (140.0 ± 7.4 vs. 44.7 ± 6.0 ng/islet) in ZDF islets (Fig. 2, B and C).

To explore the role of increased SREBP-1c expression in the functional changes observed in ZDF rat islets (29, 35, 56, 57), we overexpressed SREBP-1c DN using adenoviral transduction. Infection of either fa/+ or ZDF islets with an adenovirus encoding SREBP-1c DN increased the level of SREBP-1c mRNA by 85-fold (P < 0.001) as assessed by real-time PCR, and SREBP-1c DN protein was expressed in 30–60% of islet cells in immunostained cryosections (not shown).

SREBP-1c DN substantially lowered TG levels in ZDF (fa/fa) islets but had no significant effect on lean control islets (Fig. 2, B and C). Correspondingly, quantitative PCR revealed that peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), AP-2{gamma}, and diacylglycerol O-acyltransferase-2 (DGAT-2) mRNA levels were reduced by 70% (P < 0.001), 47% (P < 0.01), and 62% (P < 0.01), respectively, on infection of ZDF islets with SREBP-1c DN adenovirus (Fig. 3, A–C). However, unexpectedly, blockade of SREBP-1c function in ZDF islets also significantly normalized the substantial underexpression of Glut-2 mRNA in ZDF islets (Fig. 3D).

SREBP-1c DN partially rescues abnormalities in gene expression in ZDF islets. To explore in further detail the changes in gene expression in ZDF vs.fa/+ islets, mRNA profiling was performed using pancreatic islets isolated from heterozygous lean (fa/+) or ZDF rats (Fig. 4, Table 2; note that data are presented as changes in the level of a particular mRNA relative to basal in Fig. 4 and as relative cellular abundance of the message in Table 2). Of the 32,200 genes and expressed sequence tags (ESTs) represented on the arrays, 1,337 transcripts showed significant differences greater than twofold at the P < 0.01 level. Of these, 395 were downregulated and 924 were upregulated in ZDF compared with fa/+ islets. Importantly, none of the genes that have been reported to best characterize acinar pancreatic tissue (15) differed significantly between lean and ZDF islets, including carboxypeptidase, protease serine-1 or -2, amylase, elastase-3A or -3B, or chymotrypsinogen, suggesting equivalent purity of the two islet preparations. Whereas there was no significant difference in the expression of preproinsulin mRNA, glucagon and somatostatin mRNAs were reduced by 50% (P < 0.01) and 86% (P < 0.001), respectively, in ZDF vs. fa/+ islets (Fig. 4A).


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  		Table 2. mRNA expression levels in control (fa/+) and ZDF (fa/fa) is lets by biological category

 
Microarray analysis confirmed the upregulation in ZDF vs.fa/+ islets of several genes involved in lipid synthesis, including PPAR{gamma} (P < 0.001), fatty acid translocase (FAT/Cd36) (P < 0.001), DGAT (P < 0.001), and AP-2{gamma} (P < 0.001). Glycerol-3-phosphate acyltransferase (GPAT) was also increased, although this did not quite reach significance (Fig. 4B). As expected, mRNA encoding the adipocyte marker including AP-2 was also significantly increased in ZDF islets, whereas expression of carnitine palmitoyl transferase-2 (CPT-2), an important regulator of beta-oxidation, was reduced by ~50% (Fig. 4B). On the other hand, CPT-1 expression was significantly increased (Table 2).

The expression of several key "glucose-sensing" genes was significantly altered in ZDF islets (Fig. 4C). Thus Glut-2 mRNA was significantly lowered, whereas both Glut-1 (2-fold) and Glut-3 (6.4-fold) genes were upregulated (Fig. 4C), and mRNA encoding the pentose phosphate shunt enzyme glucose-6-phosphate dehydrogenase (G-6-PD) was significantly increased in ZDF islets (2.5-fold). Similarly, mRNA encoding the lactate/monocarboxylate transporter MCT1 (Slc16a1) was increased 2.8-fold (P < 0.001) and lactate dehydrogenase-A (LDH1) mRNA by 2.4-fold (P < 0.01) in ZDF islets, changes likely to diminish the mitochondrial oxidation of glucose (52). Finally, significant changes in the expression of subunits of the KATP channel were apparent, with a 40% decrease in the expression of the channel subunit Kir6.2 and a twofold increase in the muscle-specific isoform SUR2 (Fig. 4C). ZDF islets also displayed marked changes in the expression of proteins involved in the regulation of vesicle traffic and exocytosis (Fig. 4, D and E). Several of those upregulated (Fig. 4D), notably beta-actin, gelsolin, and scinderin, are involved in modeling the cortical actin network, while the microfilament-dependent motor protein myosin Vc and the microtubule-dependent motor protein kinesin 7 (and the gelsolin family member CAP-G) were also overexpressed in ZDF islets. Conversely, expression of the small GTP binding protein Rab3 (39), an inhibitor of exocytosis (30), and also Rab9b, was significantly decreased, as was expression of cdc42, an activator of actin polymerization and inhibitor of insulin release (41). By contrast, synaptotagmin V, a positive regulator of Ca2+-stimulated exocytosis in the beta-cell (33), was substantially decreased in ZDF islets, along with Rab3A-interacting molecule (RIM)-binding protein-2 (72), an effector for the RIM family of rab3 binding proteins (Fig. 4E).

Table 2 lists 57 genes whose expression was 1) significantly (P < 0.01) altered in ZDF vs. fa/+ islets and 2) by virtue of playing a likely role in either glucose or lipid metabolism, ions fluxes, or in the trafficking or exocytosis of LDCVs, deemed likely to affect secretory responses to glucose in the short term. Of these, 21 were upregulated in ZDF islets, of which 5 were decreased by SREBP-1c (Table 2). We were able to identify five distinct gene groups (groups A–E) differentially regulated in ZDF vs. fa/+ islets. Group A (Table 2) comprises genes upregulated in ZDF islets and suppressed by SREBP-1c DN in ZDF islets that are well-defined transcriptional targets of SREBP-1c (17, 31). This group included several genes involved in fatty acid metabolism and NAD(P)H synthesis, likely contributing to enhanced TG deposition, as well as annexin 1A, a positive regulator of exocytosis (43), and secretoglobin 1A (9), involved in the constitutive release of lipids. Group B (Table 2) included genes whose expression was diminished in ZDF vs. fa/+ islets but unaffected by SREBP-1c inhibition. Important members of this group were Kir6.2, rab3C and -D, and the mitochondrial ATP synthase Fo subunit c. Decreased ATP synthase activity is predicted to reduce ATP levels in ZDF islets, a feature of T2D human islets (5). Interestingly, the vacuolar proton-pumping ATPase subunit H was significantly downregulated in ZDF islets, a change not rescued by SREBP-1c inhibition. Failure of insulin-containing secretory granules to adequately acidify may thus contribute both to delayed processing of insulin and also to defective exocytosis in ZDF islets (60). In microarray analysis, the rescue by SREBP DN of Glut-2 expression in ZDF rat islets (35) fell just outside statistical significance (Table 2).

Group C comprised genes whose repression in ZDF islets might, at least in part, be due to overexpression of SREBP-1c and elevated TG levels (Table 2). Thus group C genes were significantly diminished in ZDF vs. fa/+ islets, and these changes were substantially reversed in SREBP-1c DN-treated islets. The voltage-dependent Ca2+ channel subunit-{alpha}1D previously shown to be suppressed in prediabetic and diabetic ZDF islets (51, 62) and the beta3-subunit of this channel were included in this group. Interestingly, treatment of fa/+ islets with SREBP-1c DN significantly elevated the expression of these channel subunits, implicating SREBP-1c as a repressor of these genes even under basal conditions (Table 2). The vacuolar proton-pumping ATPase Vo subunit also fell into this category, as did Ca2+-dependent activator for exocytosis (CAPS) (73) and secretogranin-3 (45), whose diminished expression may inhibit exocytosis and stabilization of the vesicle core, respectively. Because SREBP-1c is not thought to act as a direct transcriptional repressor, changes in the expression of genes in this group seem more likely to be the result of enhanced TG or fatty acid levels.

Group D (Table 2) includes those genes whose expression was decreased in ZDF vs. fa/+ islets and was further decreased by SREBP-1c inactivation. This group includes syntaxin 7 (10) and secretogranin-2, important regulators of vesicle maturation and exocytosis. Finally, group E comprises genes including MCT1 (slc16a1) and LDH1 that were upregulated in ZDF vs. fa/+ islets and either unaffected or further activated by SREBP-1c inhibition.

SREBP-1c DN rescues basal but not glucose-stimulated insulin release in ZDF islets. We next determined whether SREBP-1c DN overexpression might affect GSIS in ZDF islets. As shown in Fig. 5A, SREBP-1c DN had no effect on insulin release from control fa/+ islets at either low or stimulatory glucose concentrations, in line with normal GSIS from mice deleted for the SREBP-1 gene (61). However, in ZDF islets, the elevated insulin release observed at basal glucose was reduced to that of lean control islets (P < 0.02), while the fold induction of secretion by glucose (~2.5-fold) was preserved.

As previously reported (62), total insulin content was significantly lower in ZDF islets compared with lean controls (14.6 vs. 11.1 ng/islet, P < 0.001; Fig. 5B), possibly reflecting elevated basal secretion. A tendency toward enhanced basal secretion (Fig. 5A) may also explain the lowered insulin content of SREBP-1c DN-treated lean islets vs. null virus-affected islets (Fig. 5B).

Expression of beta-cell-specific and other islet-specific transcription factors is altered in ZDF vs. fa/+ islets. The above results indicated that, while SREBP-1c DN at least partially rescued the defective expression of several important glucose-sensing genes in ZDF islets (e.g., Glut-2), several others (e.g., Rab3, Rab9) were unaltered (Table 2), presumably explaining, at least in part, the failure of SREBP-1c DN to rescue effectively the loss of normal GSIS. To determine the extent to which this failure of SREBP-1c DN to rescue a more complete subset of genes differentially expressed in ZDF islets was likely to affect glucose metabolism and/or vesicle trafficking, we next analyzed changes in levels of key beta-cell transcription factors under each condition. While the levels of mRNAs encoding several beta-cell-restricted factors including Nkx6.1, Nkx6.2, and Pax4 were not different in fa/+ and ZDF islets, levels of those encoding pancreatic duodenum homeobox-1 (Pdx-1) (42) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) (44) were significantly (by both t-test and Q-test) less abundant in ZDF islets than in fa/+ islets (Fig. 6). In the case of both of these genes, introduction of SREBP-1c DN substantially reversed the decrease observed in ZDF islets while having no effect on the levels of either mRNA in fa/+ islets. Possibly contributing to the decrease in preproglucagon expression in ZDF islets, expression of the homeodomain factor Pax6, implicated in {alpha}-cell development but expressed throughout the islet in the adult mouse (27) and also involved in the regulation of beta-cell genes (55), was significantly diminished in ZDF islets to about the same extent as Pdx-1 and MafA, and was barely affected by SREBP-1c DN. Similarly, aristaless-related homeobox (Arx), required for {alpha}-cell development and involved in repressing beta- and {delta}-cell formation (13), was very substantially (>13-fold) diminished in ZDF islets and was not significantly affected by SREBP-1c DN expression in either fa/+ or ZDF islets.


Figure 6
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  		Fig. 6. Expression of islet-specific genes in fa/+ and ZDF islets analyzed by oligonucleotide microarray. Details of incubations and analysis are given in MATERIALS AND METHODS and in the legend to Table 2. Data are means ± SD in each case. Difference between ZDF and fa/+ islets (t-test): *P < 0.05, **P < 0.01, ***P < 0.001. Effects of SREBP-1c DN on ZDF islets: #P < 0.05 or ##P < 0.01. Where significance is shown, P < 0.01 by Q-test (see MATERIALS AND METHODS).

 
Expression of transcription factors implicated in beta-cell dysfunction is altered in ZDF vs. fa/+ islets. The above analysis revealed that SREBP-1c DN failed to efficiently rescue the expression of at least two transcription factors likely to control the expression of important downstream genes in the beta- and other islet cells, likely contributing to the inability of SREBP-1c DN to efficiently rescue GSIS in ZDF islets. To further explore the basis for defective GSIS in ZDF islets, and its unresponsiveness to SREBP-1c DN, we determined whether the expression of non-islet-restricted transcription factors, previously implicated in beta-cell dysfunction, was altered in ZDF islets. No differences were apparent in the expression of hepatocyte nuclear factor-3{alpha} (HNF3{alpha}; also termed Forkhead box a1, Foxa1) or HNF4{alpha} (implicated in maturity diabetes of the young, MODY1) (74) between ZDF and fa/+ islets. By contrast, HNF-1{alpha} (mutated in MODY3) (75) and HNF3beta, also termed Foxa2, were both downregulated in ZDF rat islets, and these changes were aggravated or unaffected, respectively, by SREBP-1c DN overexpression (Fig. 7).


Figure 7
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  		Fig. 7. Expression of genes implicated in beta-cell dysfunction in fa/+ and ZDF islets analyzed by oligonucleotide microarray. Details of incubations and analysis are given in MATERIALS AND METHODS section and in the legend to Table 2. Difference between ZDF and fa/+ islets (t-test): *P < 0.05, **P < 0.01, ***P < 0.001. Effects of SREBP-1c DN on ZDF islets: #P < 0.05. Where significance is shown, P < 0.01 by Q-test (see MATERIALS AND METHODS).

 
Inducible cyclic AMP early repressor (ICER), a splice variant of the cyclic AMP response element modulator (CREM) family, has previously been shown to be upregulated in islets from the Goto-Kakazaki rat (34), in glucagon-treated islets (32), and in clonal beta-cells incubated at elevated glucose concentrations (1), and inhibits the expression of the preproinsulin and other beta-cell genes (34). ICER expression was significantly downregulated in ZDF islets, a change only marginally affected by the introduction of SREBP-1c DN. Likewise, expression of transcription factor 7-like 2, TCF7L2 (formerly termed TCF-4), a transcription factor recently linked to increased susceptibility to T2D in humans (22), was increased in ZDF islets, a change again unaffected by blockade of SREBP-1c.


   DISCUSSION
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Islets from ZDF rats have previously been shown to be fat-laden, a change that may lead to the accumulation of nonoxidative metabolites including ceramide, loss of GSIS, and eventually beta-cell loss by apoptosis (67). Correspondingly, overexpression of SREBP-1c in ZDF islets has been proposed to be instrumental in this defect, since lowering of islet SREBP-1c levels in response to hyperleptinemia or troglitazone treatment lowered SREBP-1c expression and corrected defective insulin secretion (36). Nevertheless, the role of SREBP and of TG accumulation in the secretory deficiencies has been contested. For example, the toxicity of individual fatty acids was found to be inversely related to their capacity to induce TG deposition (12), and decreases in TG content achieved in lipid-cultured INS-1 832/13 cells or ZDF islets failed to enhance GSIS (7). However, the latter study (7) involved blockade of malonyl-CoA synthesis, a potentially important signaling step in insulin secretion (49), complicating the interpretation of these results.

As an alternative approach to addressing the role of TG accumulation in beta-cell secretory failure, we overexpressed here a SREBP-1c DN, known to be upregulated in ZDF islets (36). Prediabetic ZDF islets were deployed to minimize the potentially confounding effects of long-term exposure to elevated glucose levels. By monitoring the key functional parameters, we also sought to provide mechanistic insights into defective insulin release in the ZDF model.

We demonstrate that the secretory dysfunction of ZDF islets is associated with changes in the expression of a wide range of genes, of which a subset, implicated in glucose metabolism, ion fluxes, and/or secretory vesicle dynamics, is likely to play a role in the acute regulation of insulin release (Table 2). We suspect that only a minority of the affected genes are likely to be regulated directly by SREBP-1c binding to the corresponding promoters (Table 2, group A), whereas others (group C) may be regulated indirectly as a result of changes in cellular lipid content. In the majority of cases (groups B, D, E), changes in gene expression in ZDF islets appear to be unrelated to either parameter, and the mechanisms through which these genes in these groups are up- or downregulated in ZDF islets are unclear. Perhaps surprisingly, and despite an increase in SREBP-1c levels (~1.6-fold compared with fa/+ islets), ZDF islets did not express significantly higher levels of several SREBP-1c target genes, including fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1 (31), than lean rat islets, perhaps reflecting a role for other lipogenic transcription factors such as carbohydrate response element binding protein (71) in regulating the expression of these genes in beta-cells. Moreover, because the expression of CPT-1, an activity with a high control strength for beta-oxidation of fatty acids, was increased in ZDF lean islets, it is conceivable that both fatty acid oxidation and the esterification of fatty acids (although not necessarily fatty acid synthesis; see above) may be increased in parallel. However, a larger increase in esterification, possibly of exogenous fatty acids (whose uptake may be facilitated by the increase in the expression of Cd36; Table 2), is likely to result ultimately in the net accumulation of TG. It should be stressed that the balance between fatty acid oxidation and synthesis and the balance between esterification and desterification are subject to tight control of enzyme activity posttranslationally via the reversible phosphorylation of acetyl-CoA carboxylase and TG lipase, respectively, and are thus unlikely solely to reflect changes in gene expression.

Abrogation of SREBP-1c function failed to normalize GSIS in ZDF islets, despite changing the expression of a substantial proportion of the genes affected. This result was somewhat unexpected, given that decreases in islet SREBP-1c in response to treatment with thiazolidenediones are associated with improved GSIS (28, 36). What other factors may underlie the failure of SREBP-1c inactivation to significantly improve defective GSIS from ZDF islets, despite correcting the expression of several genes pivotal to the sensing of glucose? Although there is likely to be an important role for lipid products in stimulation-secretion coupling in the beta-cell (49), we consider a negative effect of SREBP-Ic deletion to impede the production of lipid-derived coupling factors unlikely, since SREBP-1c function is dispensable for GSIS, as demonstrated 1) by SREBP1 gene inactivation in mice (61) and 2) by the absence of any effect of SREBP-1c DN on GSIS from lean (fa/+) rat islets (Fig. 5A). On the other hand, it cannot be excluded that signaling by lipid-derived messengers assumes a greater importance in ZDF rat-derived beta-cells than in beta-cells from lean animals, such that beneficial effects of SREBP-1c inactivation (e.g., on Glut-2 expression) are countered by less efficient production of lipidic signaling molecules.

To explore in more detail the possible reasons underlying the inability of SREBP-1c DN to rescue GSIS, we analyzed here the expression of several key beta-cell transcription factors. This revealed that SREBP-1c DN reversed the changes in some (Pdx-1, MafA) but by no means all islet-specific (e.g., Pax6, Arx) transcription factor mRNA levels and also left the expression of several transcription factors implicated in beta-cell failure in other models of T2D (HNF-1{alpha}, ICER, TCF7L2; Figs. 6 and 7) unaffected in ZDF islets. Interestingly, in demonstrating significant (Pax6) or very substantial (Arx) changes in the levels of transcription factors that are important in maintaining the {alpha}-cell phenotype, these studies also provide a basis for the significant decrease in glucagon gene expression. Moreover, the simultaneous reduction in both Pdx-1 and Pax6 levels may contribute to the particularly dramatic reduction in somatostatin gene expression, given the cooperative action of these factors in the regulation of the somatatostatin gene upstream enhancer (3). We would note that the above changes in transcription factor levels are unlikely simply to reflect a "dilution" of the {alpha}- (and {delta}-) cell complement within ZDF islets by an expanded beta-cell mass, given 1) the absence of significant changes in preproinsulin mRNA levels (Fig. 4) and reductions in Pdx-1 and MafA mRNA levels (Fig. 6) and total insulin levels (Fig. 5) and 2) the absence of substantial changes in islet size (Fig. 2C).

We also describe both similarities and differences with changes in gene expression recently reported in islets recovered from human T2D (23). Thus, in human T2D (23), there were clear reductions of genes involved in signaling by insulin/IGF-I (insulin receptor, IRS-2, Akt2) that were not detected here in the rodent model. (Note, however, that the levels of mRNA encoding IRS-2 apparent in the microarrays here were <10 under all conditions and thus fell below the cutoff for further analysis; see MATERIALS AND METHODS. Thus we cannot exclude that a difference in IRS-2 mRNA levels exists between ZDF and lean rat islets.) Conversely, decreases in Pdx-1, MafA, HNF1{alpha}, HNF3beta (Foxa2), and Glut-2 expression were clearly evident in ZDF vs. control islets, while no changes in Glut-2 mRNA levels were evident in diabetic vs. control human islets (23). These differences may reflect a more important role for SREBP-1c and islet lipid accumulation in the rodent model compared with common forms of human T2D.

Which of the genes likely to regulate glucose signaling and vesicle behavior acutely might play the most important role in the loss of normal GSIS from ZDF rat islets? Previous studies (62) have reported changes in the expression of potentially important candidate genes in both prediabetic and diabetic ZDF islets. These included L-type voltage-gated Ca2+ channel (VGCC) subunits, the G protein-coupled inwardly rectifying K+ channel GIRK1, inositol 1,4,5-trisphosphate somatostatin receptors, and sarco(endo)endoplasmic reticulum Ca2+ ATPase-3 (SERCA3). Among these, only the VGCC subunits-{alpha}1D (Cav1.3), -beta2, and -beta3 were found to differ here between ZDF and control islets. Perhaps surprisingly, and in contrast to reported abnormalities in glucose- or K+-channel opener-mediated [Ca2+]i increases in older (12–14 wk) diabetic ZDF animals (51), the reduction in VGCC channel expression did not result in any change in the [Ca2+]i increase observed in response to cell depolarization in prediabetic animals (Fig. 1). There are several likely and mutually inclusive explanations for this result. First, changes in VGCC subunit mRNA level may not necessarily lead to proportional alterations at the protein level over the time frame of the present experiments. Second, the impact of any changes that do occur in the quantity of the expressed channels may be dampened if other limiting factors, such as the number of associated regulatory proteins including calmodulin (24), are unchanged. Finally, it should be stressed that L-type VGCCs are not the only Ca2+ channels activated in beta-cells by cell depolarization (40). Thus deletion of {alpha}1c-subunits by Cre-LoxP recombination in mice has no effect on glucose-induced [Ca2+]i oscillations despite strongly inhibiting glucose-induced insulin secretion (54), whereas deletion of {alpha}1D-subunits has little effect on either [Ca2+]i oscillations or insulin secretion (6).

By demonstrating a selective diminution of glucose-, but not depolarization-induced [Ca2+] increases in islets from prediabetic ZDF rats, we provide evidence that the dysregulation of insulin secretion at this stage of disease progression is unlikely to be principally the result of changes in the expression of voltage-gated Ca2+ channels. Instead, suppression of glucose transport and mitochondrial ATP synthesis, and lowered levels of the KATP channel subunit Kir6.2, may be involved. Thus we demonstrate substantial changes in the expression of KATP channel subunits (25) in prediabetic ZDF rats, including a small decrease in Kir6.2 mRNAs and a more substantial increase in SUR1 mRNA. These changes may be related to the observed decrease in HNF3beta/Foxa2 expression, a known regulator of the KATP channel subunit genes (68). However, they were apparently not sufficient to lead to a decrease in resting beta-cell membrane potential and Ca2+ influx at low glucose concentrations, given the essentially indistinguishable basal [Ca2+]i in ZDF and lean islets. Thus other changes must underlie the enhancement of basal insulin release from ZDF islets. One possibility is a change in basal dense core vesicle mobility or fusogenic potential (65). Unfortunately, we encountered significant technical difficulties in imaging vesicle behavior at the single cell level in ZDF rat-derived islet cells, including the challenge of obtaining sufficient numbers of these cells to perform such experiments. Although our studies did allow us to conclude that the number of productive exocytotic events was likely to be less in these than in control islets (consistent with measurements of insulin release at the population level), precise quantitation of vesicle diffusion coefficients proved difficult. Moreover, the impact of the SREBP-1c DN adenovirus on vesicle dynamics in ZDF islets could not readily be tested because of the interference of the GFP tag coexpressed in the viral genome, and necessary to unambiguously define infected cells, with the fluorescence of the vesicle marker NPY.Venus. More comprehensive studies will therefore be necessary to provide an accurate picture of vesicle behavior in ZDF vs. lean islets and the impact of SREBP-1c inhibition on this process. Other possibilities that may contribute to enhanced basal insulin secretion include the dramatic (86%) decrease in somatostatin gene expression, as well as the decreased expression of the exocytosis inhibitors Rab3 and Rab9 and cdc42, or upregulation of annexin 1A.

In conclusion, we demonstrate that defective GSIS in ZDF islets is associated with changes in the expression of a wide range of genes. These include both key transcription factors implicated in the global control of beta-cell (and other islet cell) gene expression and a substantial number of genes likely to be involved directly in glucose sensing and insulin release. Blockade of SREBP-1c function corrected only a subset of these changes in each case and, while reducing basal insulin release, was unable to correct the defective GSIS in ZDF islets. TG accumulation would therefore appear unable, alone, to explain the secretory defects observed in ZDF islets. Future studies, involving the systematic inactivation/knockdown of previously unsuspected targets identified here as being dysregulated in ZDF islets (see RESULTS and above), will be required to elucidate the contribution of each to the secretory phenotype.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
REFERENCES
 
We thank the Wellcome Trust (Program Grant No. 067081, to G. A. Rutter), the Medical Research Council, and Diabetes-United Kingdom for financial support. G. A. Rutter is a Wellcome Trust Research Leave Fellow.


   ACKNOWLEDGMENTS
 
We thank Jane Binz and Tracy Brainard (GlaxoSmithKline) for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. A. Rutter, Henry Wellcome Signaling Laboratories and Dept. of Biochemistry, School of Medical Sciences, Univ. 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.


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