The characteristics of the 48 non-diabetic and 12 type 2 diabetic multi-organ donors, whose pancreases were processed for islet preparation, are presented in Table 1. Pancreases were obtained and processed with the approval of the regional Ethics Committee.
Human pancreatic islets and experimental plan
Isolated pancreatic islets were prepared by collagenase digestion and density gradient purification [8, 9]. After isolation, islets were cultured free floating in M199 culture medium (Sigma-Aldrich, St Louis, MO, USA) at 5.5 mmol/l glucose concentration and studied within 3 days from isolation. Cell viability, measured by Trypan Blue exclusion, was higher than 90% in control and diabetic islets after 3 days in culture.
Insulin secretion study
Insulin secretion studies were performed as previously described [8, 9]. Following a 45 min pre-incubation period at 3.3 mmol/l glucose, groups of 30 islets of comparable size were kept at 37°C for 45 min in KRB, 0.5% (wt/wt) albumin, pH 7.4, containing 3.3 mmol/l glucose. At the end of this period, the medium was completely removed and replaced with KRB containing either 3.3, 16.7 or 3.3 mmol/l glucose plus 20 mmol/l arginine, or 3.3 mmol/l glucose plus 100 μmol/l glibenclamide. After an additional 45 min incubation period, the medium was removed. Media (500 μl aliquots from the 10 ml incubation volume) were stored at −20°C until insulin concentrations were measured by immunoradiometric assay (Pantec Forniture Biomediche, Turin, Italy).
Streptozotocin (STZ)–nicotinamide (NA)-treated male Wistar rats (2–3 months old) were administered 210 mg/kg NA i.p. (Sigma, St Louis, MO, USA) dissolved in saline, 15 min before an i.v. injection of 60 mg/kg STZ (Sigma) that had been dissolved in citrate buffer (pH 4.5) immediately before use. Control rats were injected with vehicle alone. STZ–NA-treated animals had stable hyperglycaemia (8.9–10.0 mmol/l) and they were used for the experiments 5 weeks after diabetes was induced. Pancreatic islets were isolated by the collagenase method using the procedure of pancreatic duct cannulation and density gradient purification as described elsewhere [10, 11].
GK rats were obtained from the Stockholm colony and bred as described . Inbred, normoglycaemic F344 rats were purchased from Charles River Laboratories (Wilmington, MA, USA) and maintained by sister–brother mating. Transfer of GK alleles onto the genome of F344 rats by repeated backcrossing (ten generations) established the homozygous congenic strains F344.GK-Niddm1f (NIDDM1F) and F344.GK-Niddm1i (NIDDM1I). NIDDM1F carries 0.5% of the GK genotype (8 cM), based on genetic distance, on a homozygous F344 genetic background. NIDDM1F rats display hyperglycaemia accompanied by fasting hyperinsulinaemia and increased epididymal fat, implicating insulin resistance. NIDDM1I carries 0.8% of the GK genotype (14 cM), and display hyperglycaemia and insulin secretion defects [13–15]. Backcrossing was designed to introduce mitochondrial DNA and chromosomes X plus Y from F344. The congenic strains were kept constant by sister–brother mating for several generations. To avoid effects of the oestrous cycle and other sex-specific influences, only male rats were included in this study. Rats were maintained at constant temperature and humidity in a 12 h light–dark cycle with free access to standard laboratory chow pellets and water. All experiments were approved by the local Ethics Committees. Isolated pancreatic islets were prepared from rats at 8 weeks of age after a 6 h fast (08:00–14:00 hours), by injection of a collagenase solution via the bile–pancreatic duct .
Analysis of PPARGC1A mRNA expression in pancreatic islets
Total RNA was extracted from human islets, after 3 days in culture, using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA, USA). It was quantified by absorbance at A
280 nm (ratio > 1.65) in a Perkin-Elmer spectrophotometer (Waltham, MA, USA) and its integrity was assessed after electrophoresis in 1.0% (wt/wt) agarose gels by ethidium bromide staining. Human and rat PPARGC1A mRNA expression were quantified by RT-PCR . Gene-specific probes and primer pairs for PPARGC1A (Assays-on-demands, human, Hs00173304_m1 and rat, Rn00580241_A1; Applied Biosystems, Foster City, CA, USA) were used. Each sample was run in duplicate and the transcript quantity was normalised to the mRNA level of cyclophilin A (human, 4326316E and rat Rn00574762_A1; Applied Biosystems). For each probe/primer set, a standard curve was generated, which was confirmed to increase linearly with increasing amounts of cDNA.
Downregulation of PGC-1α in human islets using silencing RNA (siRNA)
In order to test whether inhibition of PPARGC1A expression could directly modulate insulin release, isolated human islets were transfected with PPARGC1A siRNA by using Arrest-In Transfection (Open Biosystem; Celbio, Pero, Italy). This is a polymeric formulation developed and optimised for highly efficient delivery of siRNA into the nucleus of suspension cells in the presence of serum-containing medium. Pre-designed Silencer siRNAs and Silencer non-targeting siRNAs (negative control) for PPARGC1A (Ambion, Austin, TX, USA) were used. Transfection was performed according to the manufacturer’s instructions. Briefly, islets obtained from five pancreases were washed 12 times in KRB and exposed for 10 min to free Ca2+- and Mg2+-KRB to allow cell disaggregation. At the end of the incubation period, 400 islets per study point were re-suspended in 800 μl M199 medium (Sigma-Aldrich) added with adult bovine serum. siRNA (80 nmol/l) was diluted in 100 μl M199, while 20 μg Arrest-In solution (Celbio) was dissolved in 100 μl M199 and incubated for 10 min after rapid mixing to allow formation of transfection complexes. Finally, 200 μl of this solution were added into wells containing islets and incubation was allowed for 48 h in a CO2 incubator at 37°C. At the end of the incubation period well volume was doubled with M199 culture medium and samples were kept in the incubator for another 48 h, when islets function, viability, transfection efficiency (60% when measured by Polyfectamine tied to a fluorescent probe) and gene expression were evaluated.
Genomic DNA was extracted from pancreatic islets using a Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). The Gly482Ser (GGT→AGT) polymorphism of PPARGC1A was genotyped using an allelic discrimination assay performed with an ABI 7900 system (Applied Biosystems), using PCR primers: 5′-CACTTCGGTCATCCCAGTCAA-3′ (forward) and 5′-TTATCACTTTCATCTTCGCTGTCATC-3′ (reverse), and TaqMan MGB probes: Fam-5′-AGACAAGACCGGTGAA-3′ and Vic-5′-CAGACAAGACCAGTGAA-3′ [5, 17].
A sequence starting 5,000 bp upstream from the PPARGC1A translation start was used in MethPrimer (http://www.urogene.org/methprimer/index.html) to search for regions with CpG sites and PCR designs. A PPARGC1A sequence 986–746 bases upstream from the translation start including four possible DNA-methylation sites and a putative hepatic nuclear factor 1 (HNF-1) binding site, was selected and analysed for DNA methylation (Fig. 1c). Genomic DNA, isolated from pancreatic islets of nine non-diabetic and ten type 2 diabetic multi-organ donors, was treated with bisulphite using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA). Bisulphite-modified DNA was amplified by nested PCR with primers designed using MethPrimer. Primer pair 1: 5′-TAGGGTATTAGGGTTGGAATTTAATG-3′ (forward) and 5′-CCCATAACAATAAAAAATACCAACTC-3′ (reverse), and primer pair 2 (used for nested PCR): 5′-TATTTTAAGGTAGTTAGGGAGGAAA-3′ (forward) and 5′-ATAACAATAAAAAATACCAACTCCC-3′ (reverse). The PCR products were then cloned into a vector (TOPO TA Cloning Kit for Sequencing, Invitrogen, Carlsbad, CA, USA) and ten colonies from each donor were purified with a Miniprep kit (Qiagen). These individual clones were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The number of methylated sites was determined and divided by the total number of methylation sites and then multiplied by 100 to show the percentage of methylation for each donor.
Differences in PPARGC1A mRNA expression, percentage of DNA methylation and insulin secretion between the different groups studied were analysed using Student’s t test or the non-parametric Mann–Whitney test, where appropriate. Correlations were calculated using Pearson correlation coefficients for normally distributed values and Spearman correlation coefficients when normality was rejected. Log values were used in the multivariate regression analysis. Differences in expression between GK, F344, NIDDM1F and NIDDM1I rats were analysed using one-way ANOVA, followed by a Kruskal–Wallis Z test. All p values were two-tailed and p values less than 0.05 were considered significant. Statistical calculations were performed by NCSS software (NCSS Statistical Software, Kaysville, UT, USA).